Synthetic transcriptional modulators and uses thereof

ABSTRACT

Novel synthetic transcriptional modulators having at least one selected ligand linked to at least one transcriptional modulating portion are described. The transcriptional modulators of the present invention can include a ligand linked to a chemical moiety. These transcriptional modulators can be used to selectively control gene expression and to identify components of the transcriptional machinery.

BACKGROUND OF THE INVENTION

Each of the roughly 100,000 genes encoded in the human genome is subjectto individual dosage control. The systems that regulate gene expressionrespond to a wide variety of developmental and environmental stimuli,thus allowing each cell type to express a unique and characteristicsubset of its genes, and to adjust the dosage of particular geneproducts as needed. The importance of dosage control is underscored bythe fact that targeted disruption of key regulatory molecules in miceoften results in drastic phenotypic abnormalities [Johnson, R. S., etal, Cell, 71:577-586 (1992)], just as inherited or acquired defects inthe function of genetic regulatory mechanisms contribute broadly tohuman disease.

The regulatory mechanisms controlling the transcription ofprotein-coding genes by RNA polymerase II have been extensively studied.RNA polymerase II and its host of associated proteins are recruited tothe core promoter through noncovalent contacts with sequence-specificDNA binding proteins [Tjian, R. and Maniatis, T., Cell, 77:5-8 (1994);Stringer, K. F., Nature (London), 345:783-786 (1990)]. An especiallyprevalent and important subset of such proteins, known astransactivators, typically bind DNA at sites outside the core promoterand activate transcription through space contacts with components of thetranscriptional machinery, including chromatin remodeling proteins[Tjian, R. and Maniatis, T., Cell, 77:5-8 (1994); Stringer, K. F.,Nature (London), 345:783-786 (1990); Bannister, A. J. and Kouzarides,T., Nature, 384:641-643 (1996); Mizzen, C. A., et al, Cell, 87:1261-1270(1996)]. The DNA-binding and activation functions of transactivatorsgenerally reside on separate domains whose operation is portable toheterologous fusion proteins [Sadowski, I., et al, Nature, 335:563-564(1988)]. Though it is believed that activation domains are physicallyassociated with a DNA-binding domain to attain proper function, thelinkage between the two need not be covalent [Belshaw, P. J., et al.,Proc. Natl. Acad. Sci. USA, 93:4604-4607 (1996); Ho, S. H., et al,Nature (London), 382:822-826 (1996)]. In many instances, the activationdomain does not appear to contact the transcriptional machinerydirectly, but rather through the intermediacy of adapter proteins knownas coactivators [Silverman, N., et al., Proc. Natl. Acad. Sci USA,91:11005-11008 ((1994); Arany, Z., et al., Nature (London), 374:81-84(1995)].

The importance of controlled gene expression in human disease and theinformation available to date relating to the mechanisms of generegulation have fueled efforts aimed at discovering means of overridingendogenous regulatory controls or of creating new signaling circuitry incells [Belshaw, P. J., et al., Proc. Natl. Acad. Sci. USA, 93:4604-4607(1996); Ho, S. H., et al, Nature (London), 382:822-826 (1996); Rivera,V. M., et al., Nat. Med., 2:1028-1032; Spencer, D. M., et al., Science,262:1019-1024 (1993)]. Of particular interest in this regard are small,membrane-permeant molecules designed to modulate gene transcription inliving cells [Belshaw, P. J., et al., Proc. Natl. Acad. Sci. USA,93:4604-4607 (1996); Ho, S. H., et al., Nature (London), 382:822-826(1996); Rivera, V. M., et al., Nat. Med., 2:1028-1032; Spencer, D. M.,et al., Science, 262:1019-1024 (1993)]. All such efforts involvedgenetic engineering of transcriptional modulatory protein domains suchas naturally-occurring VP16. The present invention takes a significantdeparture from such art by relying upon chemical rather than biologicalmeans to harness the transcriptional machinery.

SUMMARY OF THE INVENTION

The present invention is based, at least in part, on the remarkablediscovery that small molecular weight (e.g., <5 kD), membrane-permeantcompounds are capable of acting as transcriptional modulators. Inpreferred embodiments, the compounds of the invention, also referred toherein as transcriptional modulators, include at least one selectedligand linked, e.g., covalently linked, to at least one transcriptionalmodulating portion (TMP). The TMPs of the present invention can bechemical moieties, e.g., non-peptidyl, small molecules.

Accordingly, in one aspect, this invention pertains to methods andcompositions for identifying novel transcriptional modulators. Themethod can be performed in a cell, e.g. cell-based, or in a reactionmixture, e.g., cell-free. In a cell based method, a cell is providedwhich has (a) a genetic construct encoding a chimeric protein and (b) atarget gene under the control of at least one transcriptional regulatoryelement which is recognized by the DNA-binding domain of the chimericprotein. The chimeric protein includes at least one ligand-bindingdomain (which binds to a selected ligand) and a heterologous DNA-bindingdomain. The cell is contacted with a test compound under conditionswhich allow transcription to occur and any changes in transcriptionalactivity of the target gene in the presence of the test compoundrelative to that detected in the absence of the test compound aredetected. A change in the level of transcription activity, e.g., anincrease or decrease, of the target gene detected in the presence of thetest compound relative to that detected in the absence of the testcompound indicates that the test compound is a transcriptionalmodulator.

In preferred embodiments, the test compound(s) include a selected ligandlinked to a test-transcriptional modulating portion (test-TMP). Theidentified transcriptional modulator(s) include a selected ligand linkedto a TMP.

The present invention further provides a cell-free method foridentifying a transcriptional modulator. The method involves providing areaction mixture including a chimeric protein and a target gene underthe control of at least one transcriptional regulatory element which isrecognized by the DNA-binding domain of the chimeric protein. Thereaction mixture further includes a cell-free transcription system and atest compound. Preferably, the cell-free transcription system isselected from a group consisting of a cell lysate, e.g., a HeLa cellextract, or a reconstituted protein mixture, e.g., a mixture ofcomponents of the transcriptional apparatus. The reaction mixture isprovided under conditions which allow transcription to occur and anychanges in transcriptional activity of the target gene in the presenceof the test compound relative to that observed in the absence of thetest compound are detected. In this assay, a change, e.g., an increaseor a decrease, in the level of transcriptional activity of the targetgene detected in the presence of the test compound relative to thatdetected in the absence of the test compound indicates that the testcompound is a transcriptional modulator.

In yet another aspect, the invention features a method for identifying atranscriptional modulator from a plurality of test compounds. The methodincludes: providing cells, e.g., genetically engineered cells, whichcontain (i) a genetic construct encoding a chimeric protein whichcomprises at least one ligand-binding domain and a DNA-binding domainwhich is heterologous thereto, wherein the ligand-binding domain bindsto a selected ligand; (ii) a target gene under the expression control ofat least one transcriptional regulatory element which is recognized bythe DNA-binding domain of the chimeric protein. These cells arecontacted with one or more test compounds, each of which contain theselected ligand linked to at least one of a plurality of test-TMPs; andchanges in transcription activity in the presence of the test compoundare detected relative to that detected in the absence of the testcompound. In this assay, a change, e.g., an increase or a decrease, inthe level of transcriptional activity of the target gene detected in thepresence of the test compound relative to that detected in the absenceof the test compound indicates that the test compound is atranscriptional modulator.

Another aspect of the invention pertains to a method of modulating geneexpression in a cell, e.g., a genetically-engineered cell. The methodincludes contacting the cell with a transcriptional modulator of theinvention, such that modulation of gene expression occurs. In preferredembodiments, the cell, e.g., the genetically-engineered cell, contains anucleic acid encoding a chimeric protein which comprises at least oneligand-binding domain and a DNA-binding domain heterologous thereto, andwhich binds to the transcriptional modulator. The cell can furtherinclude a target gene under the control of at least one transcriptionalregulatory element which is recognized by the DNA-binding domain of thechimeric protein. In certain embodiments, the subject methods result inactivation of transcription of the target gene. Alternatively, thesubject methods result in inhibition of expression of a target gene,e.g., a constitutively active target gene.

In preferred embodiments, the transcriptional modulator is amembrane-permeant compound which binds to the chimeric protein andactivates transcription of the target gene.

The subject method can be used on cells in culture, e.g. in vitro or exvivo. For example, cells can be cultured in vitro in culture medium andthe contacting step can be effected by adding the transcriptionalmodulator to the culture medium. Alternatively, the method can beperformed on cells present in a subject, e.g., as part of an in vivotherapeutic protocol. For in vivo methods, the cells are within asubject and the contacting is effected by administering thetranscriptional modulator to the subject. The activity of thetranscriptional modulators of the invention can be further modulated byusing a ligand which is not linked to a transcriptional modulatingdomain, which antagonizes the activity of the transcriptional modulator.

In yet another aspect, this invention pertains to a transcriptionalmodulator. The transcriptional modulator includes (i) at least oneselected ligand which binds to a ligand-binding domain of a chimericprotein, linked directly or indirectly to (ii) a transcriptionalmodulating portion (TMP). The TMP is a portion which modulatestranscription and includes one or more chemical moieties and/or one ormore proteinaceous domains.

In preferred embodiments, the transcriptional modulator is a smallmolecule, e.g., a molecule having a molecular weight less than 5 kD,preferably less than 3 kD, and even more preferably, less than 1.5 kD.Preferably, the transcriptional modulator is membrane-permeant, e.g., itis capable of passing through a cell membrane.

In preferred embodiments, the transcriptional modulator is not itselfthe product of gene transcription or translation, e.g., it is not aprotein.

In preferred embodiments, the selected ligand is linked to a TMP formingthe transcriptional modulator. Preferably, the ligand is linked to theTMP through a covalent linkage, e.g., a covalent bond, a chiral linkeror an achiral linker.

In preferred embodiments, the chimeric proteins of the invention includea ligand-binding domain which is capable of binding to at least oneselected ligand molecule and a DNA-binding domain which is capable ofbinding to a particular DNA sequence(s). The ligand-binding protein iscapable of binding with high affinity to at least one selected ligandmolecule.

Other features and advantages of the invention will be apparent from thefollowing detailed description, and from the claims.

DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1C are schematic representations of the synthesis and mechanismof activation of the transcriptional modulators (L-1 and D-1transcriptional activators).

FIG. 1A is a schematic representation of the synthesis of twotranscriptional activators which consist of a modified FK506 derivativecovalently linked to a 29-amino acid peptide of the Herpes Simplex VirusVP16 activator domain (SEQ ID NO:1). The amino acids in the VP16 peptidecan be either in the natural L stereochemical configuration (L-1), or inthe nonnatural mirror-image D configuration D stereochemistry (D-1).Abbreviations: TBS, tert-butyldimethylsilyl, Boc, t-butyloxycarbonyl.BrAc₂ O, bromoacetic acid anhydride; DMF, dimethylformamide; Me, Methyl.

FIG. 1B is a schematic representation of the structural relationshipbetween L-1 and D-1 transcriptional activators. Both artificialactivators, L-1 and D-1, contain an identical FK506 moiety (tubularstructure) attached through an achiral linker (wavy line) to either oftwo enantiomeric 29-mer activator peptides (twisted arrow).

FIG. 1C is a schematic representation of the synthetic activator servingas an intermediate between the DNA binding protein, GAL4-FKBP, and thebasal transcriptional apparatus.

FIG. 2A is a schematic representation of the target gene pG₅ FAT whichcontains five tandem 17-bp GAL4 binding sites (indicated by open boxes)positioned 23 bp upstream to the TATA box of a E4 promoter gene.

FIG. 2B is a bar graph which summarizes the quantitation of the in vitrotranscription assays using the transcriptional modulators(transcriptional activators L-1 and D-1). The template pG₅ E4T wastranscribed in a crude HeLa cell nuclear extract in the presence ofvarious added proteins and activators as indicated. The transactivator'sactivity is plotted relative to the activity of the control nuclearextract.

FIG. 3A is a schematic representation of the target plasmids and theGAL4-FKBP3 expression constructs transfected into Jurkat cells in theseexamples. The target construct (Top) G5IL2SX contains five tandem copiesof the GAL4 response element upstream of the interleukin 2 minimalpromoter and SEAP target; the GAL4-FKBP3 expression construct containsthe GAL4 DNA-binding domain fused to three tandemly repeated FKBPdomains (Bottom).

FIG. 3B is a graph depicting the results of in vivo transcriptionexperiments using the activators D-1 and L-1 in the presence of thecompetitor rapamycin (1 μM).

DETAILED DESCRIPTION OF THE INVENTION

The present invention pertains, at least in part, to the discovery ofsmall molecular weight (e.g., <5 kD), membrane-permeantnon-proteinaceous compounds which are capable of modulatingtranscription, as well as methods of using the same to regulate geneexpression, or to identify components of the transcriptional machinery.These transcriptional modulators include at least one selected ligandlinked, e.g., covalently linked, directly or indirectly to at least onetranscriptional modulating portion (TMP). The transcriptional modulatorsof the invention are capable of modulating transcription uponinteraction with a chimeric protein containing at least oneligand-binding domain which recognizes the selected ligand fuseddirectly or indirectly to at least one DNA-binding domain.

I. Methods for Identifying Novel Transcriptional Modulators

In one embodiment, the invention pertains to a method for identifying atranscriptional modulator. The method can be performed in a cell, e.g.cell-based or in a reaction mixture, e.g., cell-free. In a cell basedmethod, a cell is provided which has a genetic construct encoding achimeric protein and a target gene under the control of at least onetranscriptional regulatory element which is recognized by theDNA-binding domain of the chimeric protein. The chimeric proteinincludes at least one ligand-binding domain which binds to a selectedligand and a DNA-binding domain which is heterologous thereto. The cellis further contacted with a test compound under conditions which allowtranscription to occur and any changes in transcriptional activity ofthe target gene in the presence of the test compound relative to thatdetected in the absence of the test compound are detected. A change inthe level of transcription activity of the target gene detected in thepresence of the test compound relative to that detected in the absenceof the test compound indicates that the test compound is atranscriptional modulator. The test compound(s) include a selectedligand for the chimeric protein linked to a test-transcriptionalmodulating portion (hereinafter test-TMP). The identifiedtranscriptional modulator(s) include a selected ligand linked to a TMP.The test compounds and transcriptional modulators of the presentinvention are described in further detail below.

The present invention further provides a cell-free method foridentifying a transcriptional modulator. The method involves providing areaction mixture including a chimeric protein and a target gene underthe control of at least one transcriptional regulatory element which isrecognized by the DNA-binding domain of the chimeric protein. Thereaction mixture further includes a cell-free transcription system and atest compound. Preferably, the cell-free transcription system isselected from a group consisting of a cell lysate, e.g., a HeLa cellextract, or a reconstituted protein mixture, e.g., a mixture ofcomponents of the trasncriptional apparatus. The reaction mixture isprovided under conditions which allow transcription to occur and anychanges in transcriptional activity of the target gene in the presenceof the test compound relative to that detected in the absence of thetest compound are detected. A change in the level of transcriptionalactivity of the target gene detected in the presence of the testcompound relative to that detected in the absence of the test compoundindicates that the test compound is a transcriptional modulator.

Changes in transcriptional activity can be detected by variations in theexpression of a target gene (e.g., a reporter gene), e.g., variations inthe observed levels of mRNA, or protein product encoded by the targetgene. As used in the methods described herein, by "target gene" is meanta gene whose expression may be assayed; such genes include, withoutlimitation, genes conferring a drug resistant phenotype (e.g.,resistance to chloroamphenicol or neomycin), genes whose expressionproducts provide for calorimetric, fluorescent or luminescent detection(e.g., Green Fluorescent Protein (GFP), SEAP, β-galactosidase orluciferase), a gene whose expression product rescues an auxotrophicphenotype (e.g., LEU2, HIS3, URA3 or LYS2), or a gene encoding any cellsurface antigen for which antibodies are available (e.g., for panning).

Test Compound

The test compound can be designed to incorporate a moiety known to bindto a component of the transcription machinery or to have transcriptionalmodulating activity, or can be selected from a library of diversecompounds, e.g., based on a desired activity, e.g., random drugscreening based on a desired activity. Preferably, the test compound ofthe present invention is a small molecule having a chemical moiety,e.g., a non-peptidyl moiety as the TMP. The terms "chemical moiety" or"moiety" are intended to include synthetic and naturally-occurringnon-proteinaceous entities. For example, chemical moieties includeunsubstituted or substituted alkyl, aromatic, or heterocyclyl moietiesincluding macrolides, leptomycins and related natural products oranalogs thereof. The particular components of the test compound arediscussed in detail below in the Transcriptional Modulator and/or TestCompound section.

Libraries of Test Compounds

Novel transcriptional modulators can be identified by linking, e.g.,covalently linking, e.g., a test library to a selected ligand such thatthe test library is targeted to the transcriptional machinery. Exemplarytest libraries that can be used include combinatorial libraries.

In one embodiment, the invention provides libraries of transcriptionalmodulators. The synthesis of combinatorial libraries is well known inthe art and has been reviewed (see, e.g., E. M. Gordon et al., J. Med.Chem. (1994) 37:1385-1401; DeWitt, S. H.; Czarnik, A. W. Acc. Chem. Res.(1996) 29:114; Armstrong, R. W.; Combs, A. P.; Tempest, P. A.; Brown, S.D.; Keating, T. A. Acc. Chem. Res. (1996) 29:123; Ellman, J. A. Acc.Chem. Res. (1996) 29:132; Gordon, E. M.; Gallop, M. A.; Patel, D. V.Acc. Chem. Res. (1996) 29:144; Lowe, G. Chem. Soc. Rev. (1995) 309,Blondelle et al. Trends Anal. Chem. (1995) 14:83; Chen et al. J. Am.Chem. Soc. (1994) 116:2661; U.S. Pat. Nos. 5,359,115, 5,362,899, and5,288,514; PCT Publication Nos. WO92/10092, WO93/09668, WO91/07087,WO93/20242, WO94/08051). The subject invention includes methods forsynthesis of combinatorial libraries of transcriptional modulators. Suchlibraries can be synthesized according to a variety of methods. In oneillustrative method, a selected ligand portion is chemically linked(covalently or non-covalently) to a transcriptional modulating portion(TMP), optionally by means of a linker portion. Thus, in certainembodiments, a compound of the invention can be represented by theformula A-B-C, in which A is a selected ligand (e.g., as describedhereinbelow under the heading "Selected Ligands"); B is a direct(preferably single) bond or a linker portion; and C is a TMP. Exemplarytranscriptional modulating portions are described hereinbelow (e.g.,under the heading "Transcriptional Modulating Portions (TMP)",andinclude portions which are capable of interacting directly or indirectlywith the transcriptional machinery of a cell, or otherwise modulatestranscriptional activity, e.g., by modulating (i.e., increase ordecrease) the nuclear transport (e.g., import or export) of atranscriptional modulator, thereby modulating the effectiveconcentration of the transcriptional modulator in the cell nucleus.

Libraries of compounds of the invention can be prepared according to avariety of methods, some of which are known in the art. For example, a"split-pool" strategy can be implemented in the following way: beads ofa functionalized polymeric support are placed in a plurality of reactionvessels; a variety of polymeric supports suitable for solid-phasepeptide synthesis are known, and some are commercially available (forexamples, see, e.g., M. Bodansky "Principles of Peptide Synthesis",2ndedition, Springer-Verlag, Berlin (1993)). To each aliquot of beads isadded a solution of a different activated amino acid, and the reactionsare allow to proceed to yield a plurality of immobilized amino acids,one in each reaction vessel. The aliquots of derivatized beads are thenwashed, "pooled" (i.e., recombined), and the pool of beads is againdivided, with each aliquot being placed in a separate reaction vessel.Another activated amino acid is then added to each aliquot of beads. Thecycle of synthesis is repeated until a desired peptide length isobtained. The amino acid residues added at each synthesis cycle can berandomly selected; alternatively, amino acids can be selected to providea "biased" library, e.g., a library in which certain portions of the TMPare selected non-randomly, e.g., to provide a TMP having knownstructural similarity or homology to a known modulator of transcription.It will be appreciated that a wide variety of peptidic, peptidomimetic,or non-peptidic compounds can be readily generated in this way.

The "split-pool" strategy results in a library of peptides, e.g., TMPs,which can then be linked, covalently or non-covalently, to a selectedligand (e.g., as described herein), thereby preparing a library of testcompounds of the invention. In one embodiment, an amino acid residue(e.g., a terminal amino acid residue) of a peptidic TMP can be acysteine residue; the side-chain thiol group of the cysteine can be usedfor attachment of the peptide to a ligand (e.g., FK506 or an analog orderivative thereof). Thus, attachment of the FK506 derivative to thecombinatorial peptide library provides a library of hybridligand-peptide compounds which can then be screened for transcriptionalmodulating activity as described herein.

In another illustrative synthesis, a "diversomer library" is created bythe method of Hobbs DeWitt et al. (Proc. Natl. Acad. Sci. U.S.A. 90:6909(1993)). Other synthesis methods, including the "tea-bag" technique ofHoughten (see, e.g., Houghten et al., Nature 354:84-86 (1991)) can alsobe used to synthesize libraries of compounds according to the subjectinvention.

In certain embodiments, the invention relates to libraries of compoundswhich can modulate transcription by modulating import or export oftranscriptional modulating compounds into or out of the cell nucleus. Inthis embodiment, the TMP can be, e.g., a portion which regulates ormodulates nuclear import or export. A compound of the invention having aselected ligand portion and nuclear exporting portion can bind to afusion protein having a ligand-binding domain and promote the export ofthe fusion protein from the nucleus. (For a general discussion ofnuclear protein import, see, e.g., Gorlich, D. Curr. Opin. Cell. Biol.(1997) 9:412-419). Thus, the effective nuclear concentration of a fusionprotein having a DNA-binding domain, a ligand-binding domain and atranscriptional modulating domain can be modulated by a hybrid compoundof the invention, thereby modulating transcriptional modulatingactivity. One example of a modulator of nuclear export is leptomycin B.Thus, a library of compounds in which the TMP is a library of leptomycinanalogs or derivatives, can be prepared. Leptomycin B has a carboxylategroup which can be conveniently coupled to a ligand portion to provide ahybrid compound of the invention.

Libraries of compounds can be screened to determine whether any membersof the library have a desired activity, and, if so, to identify theactive species. Methods of screening combinatorial libraries have beendescribed (see, e.g., Gordon et al., J. Med. Chem., supra). Solublecompound libraries can be screened by affinity chromatography with anappropriate receptor to isolate ligands for the receptor, followed byidentification of the isolated ligands by conventional techniques (e.g.,mass spectrometry, NMR, and the like). Immobilized compounds can bescreened by contacting the compounds with a soluble receptor;preferably, the soluble receptor is conjugated to a label (e.g.,fluorophores, colorimetric enzymes, radioisotopes, luminescentcompounds, and the like) that can be detected to indicate ligandbinding. Alternatively, immobilized compounds can be selectivelyreleased and allowed to diffuse through a membrane to interact with areceptor. Exemplary assays useful for screening the libraries of theinvention are described below.

In one embodiment, compounds of the invention which include a ligandportion and a transcriptional modulating portion which is capable ofinteracting with the transcriptional machinery of a cell can be screenedfor transcriptional modulating activity by assaying the activity of eachcompound, e.g., by incubating the test compound with a cell or nuclearextract, e.g., in one well of a multiwell plate, such as a standard96-well microtiter plate. In this embodiment, the activity of eachindividual compound can be determined. Thus, for example, a plurality oftest compounds can be screened by incubation of each compound in a wellof a multiwell plate containing a HeLa nuclear extract (e.g., preparedas described infra) and a chimeric or fusion protein which includes aligand binding domain, to which the ligand portion of the test compoundcan bind. A well or wells having no test compound can be used as acontrol. After incubation, the activity of each test compound can bedetermined by assaying each well for a product of gene transcription.Thus, the activities of a plurality of test compounds can be determinedin parallel.

In another embodiment of a screening assay, a compound (or compounds) ofthe invention can be screened by contacting the compound with anengineered cell in vitro. For example, as described infra, a Jurkat celltransfected with a reporter gene, e.g., a secreted alkaline phosphatase(SEAP) gene under the expression control of a suitable promoter (see,e.g., PCT Application No. PCT/US94/01617) together with a DNA constructencoding a chimeric protein of this invention can be incubated with atest compound. The fusion protein can include a DNA-binding domain, anda ligand binding domain, to which the ligand portion of the testcompound can bind, as described herein. A test compound withtranscriptional activating activity will bind to the ligand-bindingdomain of the fusion protein and increase transcription of the AP geneand secretion of AP by the cell; the AP secretion readily can bedetected by known methods. Thus, the test compounds of the invention canbe rapidly screened for transcriptional activating activity. Otherreporter genes (such as luciferase or beta-galactosidase) can be used todetect transcriptional activating activity, as the ordinarily skilledartisan will appreciate.

Another embodiment relies upon the use of an auxotrophic yeast strainengineered to contain a gene which complements the auxotrophy under thecontrol of a transcription regulatory element which is recognized by theDNA-binding domain of a chimeric protein.

Another embodiment relies upon a cell (e.g., a yeast cell) lacking agene required for biosynthesis of an essential nutrient (such as, e.g.,the amino acid histidine) can be grown in a medium in which the nutrientis lacking, in the presence of a plasmid containing the gene (e.g., HIS,a histidine synthesis construct) under the control of an appropriatecontrol sequence. In the absence of a transcriptional activator, littleor no cell growth will be seen. However, in the presence of a compoundwhich has transcriptional activating activity, the cell can express therequired gene product; accordingly, cell growth can be observed when anactive compound is present.

In still another embodiment, large numbers of test compounds can besimultaneously tested for transcriptional modulating activity. Forexample, test compounds can be synthesized on solid resin beads in a"one bead-one compound" synthesis; the compounds can be immobilized onthe resin support through a photolabile linker. A plurality of beads(e.g., as many as 100,000 beads or more) can then be combined with yeastcells and sprayed into a plurality of "nano-droplets", in which eachdroplet includes a single bead (and, therefore, a single test compound).Exposure of the nano-droplets to UV light then results in cleavage ofthe compounds from the beads. The effect of the test compound on thecell (e.g., cell growth, cell death, production of gene products, andthe like) can then be measured to determine the effect of the testcompound on translation. It will be appreciated that this assay formatallows the screening of large libraries of test compounds in a rapidformat.

Combinatorial libraries of compounds can be synthesized with "tags" toencode the identity of each member of the library (see, e.g., W. C.Still et al., U.S. Pat. No. 5,565,324 and PCT Publication Nos. WO94/08051 and WO 95/28640). In general, this method features the use ofinert, but readily detectable, tags, that are attached to the solidsupport or to the compounds. When an active compound is detected (e.g.,by one of the techniques described above), the identity of the compoundis determined by identification of the unique accompanying tag. Thistagging method permits the synthesis of large libraries of compoundswhich can be identified at very low levels. Such a tagging scheme can beuseful, e.g., in the "nano-droplet" screening assay described above, toidentify compounds released from the beads.

In preferred embodiments, the libraries of transcriptional modulatorcompounds of the invention contain at least 30 compounds, morepreferably at least 100 compounds, and still more preferably at least500 compounds. In preferred embodiments, the libraries oftranscriptional modulator compounds of the invention contain fewer than10⁹ compounds, more preferably fewer than 10⁸ compounds, and still morepreferably fewer than 10⁷ compounds.

II. Methods of Modulating Gene Expression

Another aspect of the invention pertains to a method of modulating geneexpression in a cell. The method includes contacting the cell with atranscriptional modulator of the invention, such that modulation of geneexpression occurs. Preferably, the cell contains a genetic constructencoding a chimeric protein which includes at least one ligand-bindingdomain which binds to the ligand of the transcriptional modulator, fusedto a heterologous DNA-binding domain. In certain embodiments, e.g.,those involving regulated nuclear import or export, the chimeric proteincan additionally include a transcriptional modulating domain. Generally,the cell can further include a target gene under the control of at leastone transcriptional regulatory element which is recognized by theDNA-binding domain of the chimeric protein. The subject method can leadto activation of transcription of the target gene, or to inhibition ofexpression of a target gene, e.g., a constitutively active target gene.

"Cells," "genetically-engineered cells" or "recombinant cells" are artrecognized terms and the decription below applies to these cellsinterchangeably. It is understood that such terms refer not only to theparticular subject cell but to the progeny or potential progeny of sucha cell. Because certain modifications may occur in succeedinggenerations due to either mutation or environmental influences, suchprogeny may not, in fact, be identical to the parent cell, but are stillincluded within the scope of the term as used herein.

The subject method can be used on cells in culture, e.g. in vitro or exvivo, as shown in the Examples herein. For example, cells can becultured in vitro in culture medium and the contacting step can beeffected by adding the transcriptional modulator to the culture medium.Alternatively, the method can be performed on cells present in asubject, e.g., as part of an in vivo therapeutic protocol. For in vivomethods, the cell is preferably found in a subject and the contacting iseffected by administering the transcriptional modulator to the subject.As used herein, the language "subject" is intended to include human andnon-human animals. The term "non-human animals" includes allvertebrates, e.g., mammals and non-mammals, such as non-human primates,sheep, dog, cow, chickens, amphibians, reptiles, etc. In certainembodiments, the subject is a mammal, e.g., a primate, e.g., a human.

The cells used in the methods described herein may be procaryotic, butare preferably eucaryotic, including plant, yeast, worm, insect andmammalian. At present it is especially preferred that the cells bemammalian cells, particularly primate, more particularly human, but canbe associated with any animal of interest, particularly domesticatedanimals, such as equine, bovine, murine, ovine, canine, feline, etc.Among these species, various types of cells can be involved, such ashematopoietic, neural, mesenchymal, cutaneous, mucosal, stromal, muscle,spleen, reticuloendothelial, epithelial, endothelial, hepatic, kidney,gastrointestinal, pulmonary, etc. Of particular interest arehematopoietic cells, which include any of the nucleated cells which maybe involved with the lymphoid or myelomonocytic lineages. Of particularinterest are members of the T- and B-cell lineages, macrophages andmonocytes, myoblasts and fibroblasts. Also of particular interest arestem and progenitor cells, such as hematopoietic neural, stromal,muscle, hepatic, pulmonary, gastrointestinal, etc. Most preferably, thecells are muscle cells.

The cells can be autologous cells, syngeneic cells, allogenic cells andeven in some cases, xenogeneic cells. The cells may be modified bychanging the major histocompatibility complex ("MHC") profile, byinactivating b₂ -microglobulin to prevent the formation of functionalClass I MHC molecules, inactivation of Class II molecules, providing forexpression of one or more MHC molecules, enhancing or inactivatingcytotoxic capabilities by enhancing or inhibiting the expression ofgenes associated with the cytotoxic activity, or the like.

In some instances specific clones or oligoclonal cells may be ofinterest, where the cells have a particular specificity, such as T cellsand B cells having a specific antigen specificity or homing target sitespecificity.

The activity of the transcriptional modulators of the invention can befurther modulated by using a free monomeric ligand, e.g., a ligand whichis not linked to a TMP, which antagonizes the activity of thetranscriptional modulator. These free ligands may competitively interactwith a ligand-binding protein to decrease or eliminate itstranscriptional modulatory activity. Thus, if one wishes to rapidlyterminate the effect of cellular activation, a cell exposed to atranscriptional modulator of the invention can be contacted with thefree ligand to reduce or eliminate the transcriptional activity of themodulator.

Target Genes

As used herein, the term "target gene" refers to a gene which includes atranscriptional initiation region having a target DNA sequence(s) orresponsive element which is recognized by the DNA-binding domain of achimeric protein, so as to be responsive to signal initiation from theTMP. In certain embodiments, signal initiation leads to transcriptionactivation and expression of one or more target genes. Alternatively,signal initiation can lead to inhibition of transcription of an activetarget gene, e.g., a constitutively active target gene. The target genesmay be an endogenous or exogenous gene. By "exogenous gene" is meant agene which is not otherwise normally expressed by the cell, e.g. becauseof the nature of the cell, because of a genetic defect of the cell,because the gene is from a different species or is a mutated orsynthetic gene, or the like. Such gene can encode a protein, antisensemolecule, ribozyme etc., or can be a DNA sequence comprising anexpression control sequence linked or to be linked to an endogenous genewith which the expression control sequence is not normally associated.Conversely, an "endogenous gene" refers to a gene which is normallyexpressed in a given cell.

Accordingly, the constructs containing the target genes can have a"transcriptional regulatory sequence". This term refers to one or moreresponsive element(s) in the 5' region which is operably linked to atarget gene. By "operably linked" is meant that a gene and a regulatorysequence(s) are connected in such a way as to permit gene expressionwhen the appropriate molecules (e.g., chimeric proteins bound tosynthetic transcriptional modulator or a target gene) are bound to theregulatory sequences. The responsive elements(s) is recognized by theDNA-binding domain of the chimeric receptor protein and responds to theinteraction between the transcriptional modulator and the chimericprotein. In preferred embodiments, transcription of a target gene isunder the control of a promoter sequence (or other transcriptionalregulatory sequence) which controls the expression of the recombinantgene in a cell-type in which expression is intended. It will beunderstood that the recombinant gene can be under the control oftranscriptional regulatory sequences which are the same or which aredifferent from those sequences which control transcription of thenaturally-occurring form of the target gene.

The expression construct will therefore have at its 5' end in thedirection of transcription, the responsive element and the promotersequence which allows for induced transcription initiation of a targetgene of interest, usually a therapeutic gene. The transcriptionaltermination region is not as important, and can be used to enhance thelifetime of or make short half-lived mRNA by inserting AU sequenceswhich serve to reduce the stability of the mRNA and, therefore, limitthe period of action of the protein. Any region can be employed whichprovides for the necessary transcriptional termination, and asappropriate, translational termination.

The responsive element can be a single recognition sequence or multiplerecognition sequences.

Homologous recombination can also be used to replace endogenoustranscriptional control sequences with a transcriptional control elementwhich is recognized by a chimeric protein of this invention.

A wide variety of genes can be employed as the target gene, includinggenes that encode a protein of interest or an antisense sequence ofinterest or a ribozyme of interest. The target gene can be any sequenceof interest which provides a desired phenotype. The target gene canexpress a surface membrane protein, a secreted protein, a cytoplasmicprotein, or there can be a plurality of target genes which can expressdifferent types of products. The target gene may be an antisensesequence which can modulate a particular pathway by inhibiting atranscriptional regulation protein or turn on a particular pathway byinhibiting the translation of an inhibitor of the pathway. The targetgene can encode a ribozyme which may modulate a particular pathway byinterfering, at the RNA level, with the expression of a relevanttranscriptional modulator or with the expression of an inhibitor of aparticular pathway. The proteins which are expressed, singly or incombination, can involve homing, cytotoxicity, proliferation, immuneresponse, inflammatory response, clotting or dissolving of clots,hormonal regulation, or the like. The proteins expressed could benaturally-occurring, mutants of naturally-occurring proteins, uniquesequences, or combinations thereof. Particularly preferred examples oftarget genes encode a protein selected from a growth or adifferentiation factor, a protein involved in clotting or thrombolyis, aprotein involved in promoting or inhibiting vascularization, a proteininvolved in metabolic regulation, an enzyme, or a tumor suppressor.

The target gene can be any gene which is secreted by a cell, so that theencoded product can be made available at will, whenever desired orneeded by the host. Various secreted products include hormones, such asendostatin, angiostatin, insulin, human growth hormone, glucagon,pituitary releasing factor, ACTH, melanotropin, relaxin, etc.;differentiation or growth factors, such as EGF, IGF-1, TGF-α, -β, PDGF,G-CSF, M-CSF, GM-CSF, FGF, erythropoietin, neurotrophins, e.g., nervegrowth factor (NGF), brain-derived neurotrophic facto (BDNF),neurotrophin-3 (NT-3), megakaryocytic stimulating and growth factors,etc.; interleukins, such as IL-1 to -13; TNF-α and -β, etc.; solublereceptors for TNF or IL-1, or receptor antagonists therefor; enzymes,such as tissue plasminogen activator, members of the complement cascade,perforins, superoxide dismutase, coagulation factors, antithrombin-III,Factor VIIIc, Factor VIIIvW, a-anti-trypsin, protein C, protein S,endorphins, dynorphin, bone morphogenetic protein, CFTR, etc.; andleptin and leptin receptor molecules.

The target gene can be any gene which is naturally a surface membraneprotein or made so by introducing an appropriate signal peptide andtransmembrane sequence. Various proteins include homing receptors, e.g.L-selectin (Mel-14), blood-related proteins, particularly having akringle structure, e.g. Factor VIIIc, Factor VIIIvW, hematopoietic cellmarkers, e.g. CD3, CD4, CD8, B cell receptor, TCR subunits α, β, γ, δ,CD10, CD19, CD28, CD33, CD38, CD41, etc., receptors, such as theinterleukin receptors IL-2R, IL-4R, etc., channel proteins, for influxor efflux of ions, e.g. H⁺, Ca⁺², K⁺, Na⁺, Cl⁻, etc., and the like;CFTR, tyrosine activation motif, z activation protein, etc.

Proteins may be modified for transport to a vesicle for exocytosis. Byadding the sequence from a protein which is directed to vesicles, wherethe sequence is modified proximal to one or the other terminus, orsituated in an analogous position to the protein source, the modifiedprotein will be directed to the Golgi apparatus for packaging in avesicle. This process in conjunction with the presence of the chimericproteins for exocytosis allows for rapid transfer of the proteins to theextracellular medium and a relatively high localized concentration.

Also, intracellular proteins can be of interest, such as proteins inmetabolic pathways, modulatory proteins, steroid receptors,transcription factors, etc., particularly depending upon the nature ofthe host cell. Some of the proteins indicated above can also serve asintracellular proteins.

The following are a few illustrations of different target genes. InT-cells, one may wish to introduce genes encoding one or both chains ofa T-cell receptor. For B-cells, one could provide the heavy and lightchains for an immunoglobulin for secretion. For cutaneous cells, e.g.keratinocytes, particularly stem cells keratinocytes, one could providefor infectious protection, by secreting α-, β- or -γ interferon,antichemotactic factors, proteases specific for bacterial cell wallproteins, etc.

In addition to providing for expression of a target gene havingtherapeutic value, there will be many situations where one may wish todirect a cell to a particular site. The site can include anatomicalsites, such as lymph nodes, mucosal tissue, skin, synovium, lung orother internal organs or functional sites, such as clots, injured sites,sites of surgical manipulation, inflammation, infection, etc. Byproviding for expression of surface membrane proteins which will directthe host cell to the particular site by providing for binding at thehost target site to a naturally-occurring epitope, localizedconcentrations of a secreted product can be achieved. Proteins ofinterest include homing receptors, e.g. L-selectin, GMP140, CLAM-1,etc., or addressins, e.g. ELAM-1, PNAd, LNAd, etc., clot bindingproteins, or cell surface proteins that respond to localized gradientsof chemotactic factors. There are numerous situations where one wouldwish to direct cells to a particular site, where release of atherapeutic product could be of great value.

In many situations one may wish to be able to kill the modified cells,where one wishes to terminate the treatment, the cells becomeneoplastic, in research where the absence of the cells after theirpresence is of interest, or other event. For this purpose one canprovide for the expression of the Fas antigen or TNF receptor fused to abinding moiety. (Watanable-Fukunaga et al. Nature (1992) 356, 314-317)In the original modification, one can provide for constitutiveexpression of such constructs, so that the modified cells have suchproteins on their surface or present in their cytoplasm. Alternatively,one can provide for controlled expression, where the same or differentligand can initiate expression and initiate apoptosis. By providing forthe cytoplasmic portions of the Fas antigen or TNF receptor in thecytoplasm joined to binding regions different from the binding regionsassociated with expression of a target gene of interest, one can killthe modified cells under controlled conditions.

III. Transcriptional Modulators and/or Test Compounds

The present invention further pertains to transcriptional modulators.The transcriptional modulator has (i) at least one selected ligand whichbinds to a ligand-binding domain of a chimeric protein linked to (ii) atranscriptional modulating portion (TMP) forming the transcriptionalmodulator. The transcriptional modulator can be a "small molecule" inthat it includes a molecule having a molecular weight of less than about5 kD. The transcriptional regulator can be membrane permeant, e.g.,capable of passing through a cell membrane.

The present invention also includes combinations of transcriptionalmodulators. The combinations of transcriptional modulators can be usedin the methods described herein. The transcriptional modulators used inthe combinations can affect the activity of each other. For example, thecombination of transcriptional modulators can act synergistically,additively, or even in some instances counteract each other to someextent. The present invention also includes membrane-permeantformulations. In membrane-permeant formulations, at least onetranscriptional modulator is combined with an agent which enhancesmembrane permeability of the transcriptional modulator, Examples of suchagents include a detergent, such as digitonin.

It should be understood that the test compound(s) described above arebeing screened for the ability to serve as transcriptional modulators ofthe present invention. In the context of the present invention, the testcompound is a selected ligand linked to a test-TMP which is a TMP beingtested for its ability to modulate transcription. The definitions setforth below with respect to the selected ligand and/or TMP also apply tothe test compound.

The transcriptional modulator can have the following formula:

    (Ligand).sub.n -Linker-(TMP).sub.n

wherein n is an integer from 1 to about 4. A preferred transcriptionalmodulator has the formula: (Ligand)₁ -Linker-(TMP)₁. In transcriptionalmodulators where n>than 1, either one of the ligand or the TMP may bethe same or different molecules. Exemplary ligands and transcriptionalmodulators are described in detail below.

The language "small molecule" is intended to include a molecule which isnot itself the product of gene transcription of translation, i.e., it isnot a protein, RNA or DNA. Preferably, a "small molecule" has amolecular weight of less than 5 kD, preferably less than 3 kD, and evenmore preferably, less than 1.5 kD.

Preferred transcriptional modulators are membrane-permeant. As usedherein, the term "membrane-permeant transcriptional modulators" isintended to include molecules which are capable of translocating acrossthe membrane of a cell in a form that allows the molecule to perform itsintended function. Accordingly, the size and the hydrophobicity of themembrane-permeant transcriptional modulators can be determined such thattransfer across the cell membrane is effected. The cells may beprocaryotic, but are preferably eucaryotic, and most preferablymammalian cells. The membrane can consist of primarily a double layer oflipid molecules and associated proteins that enclose all cells, and, ineucaryotic cells, many organelles. As described in detail herein,various ligands and TMPs are hydrophobic or can be made so byappropriate modification with lipophilic groups. Particularly, thecovalent linkage bridging the ligand and transcriptional modulatormoiety can serve to enhance the lipophilicity of the transcriptionalmodulators by providing aliphatic side chains of from about 12 to 24carbon atoms. Alternatively, one or more groups can be provided whichwill enhance transport across the membrane, e.g., without endosomeformation.

Applicable and readily observable or measurable criteria for selecting atranscriptional modulator include: (A) the transcriptional modulator isphysiologically acceptable (i.e., lacks undue toxicity towards the cellor animal for which it is to be used), (B) the transcriptional modulatorhas a reasonable therapeutic dosage range, (C) desirably (forapplications in whole animals, including gene therapy applications), itcan be taken orally (is stable in the gastrointestinal system andabsorbed into the vascular system), (D) it can cross the cellular andother membranes, as necessary.

Selected Ligands

The terms "selected ligand" or "ligand" are art-recognized and areintended to include a molecule that binds to a protein or othermolecule, e.g., through a ligand-binding domain of said protein. Atranscriptional modulator of the present invention comprises a ligandmoiety which brings the TMP in close proximity to the target gene.Measurable criteria for selecting a ligand include a ligand which bindsto a ligand-binding domain (also referred to herein as a receptor) withreasonable affinity for the desired application. A first desirablecriterion is that the compound is relatively physiologically inert, butfor its binding to the ligand-binding domain. The less the ligand bindsto native proteins, the better the response will normally be. For themost part, the ligands can be non-peptide and non-nucleic acid.

There are a variety of naturally-occurring receptors for smallnon-proteinaceous organic molecules, which small organic moleculesfulfill the above criteria. Substantial modifications of these compoundsare permitted, so long as the binding capability is retained and withthe desired specificity. Exemplary compounds include macrocyclicmolecules, e.g., macrolides. Suitable binding affinities will bereflected in Kd values well below about 10⁻⁴ M, preferably below about10⁻⁶ M, more preferably below about 10⁻⁷ M, even more preferably belowabout 10⁻⁸ M, and in some embodiments below about 10⁻⁹ M.

Preferred ligands are membrane permeant, i.e., ligands are selected suchas to be able to translocate across the membrane. Various ligands arehydrophobic or can be made so by appropriate modification withlipophilic groups. Alternatively, one or more groups can be providedwhich will enhance transport across the membrane, desirably withoutendosome formation.

Examples of ligands include FK506, FK520, rapamycin, cyclosporin A,tetracycline, steroid, e.g., estrogen, ecdysone, or glucocorticoid,vitamin D, and derivatives thereof, which retain their bindingcapability to the natural or mutagenized ligand-binding domain. Thepresent invention takes advantage of the high affinity interactioninvolving the ligand and its natural receptor(s), e.g., the interactionbetween FK506 and an immunophilin receptor, e.g., FKBP12; cyclosporin Aand a cyclophilin receptor; a steroid and a steroid receptor, e.g.,estrogen with an estrogen receptor, ecdysone with an ecdysone receptor,or glucocorticoid with a glucocorticoid receptor; tetracycline with thetetracycline receptor; vitamin D with the vitamin D receptor; rapamycinwith FKBP which may further associate with a large mammalian proteintermed FRAP, and the like (See e.g., WO 96/41865).

Preferably, one uses a derivative of such a ligand which hassubstantially lower binding affinity for the ligand's native receptor ascompared to binding to genetically engineered variants thereof which canbe used in the chimeric proteins. As describe in detail below, inaddition to modifying the ligand, it is desirable to change the bindingprotein to accommodate the change in the ligand. For example, one canprepare modified ligands that will fail to bind appreciably to theirwildtype receptors (e.g., FKBP12) due to the presence of substituents("bumps") on the reagents that sterically clash with sidechain residuesin the receptor's binding pocket. One may also make correspondingreceptors that contain mutations at the interfering residues("compensatory mutations") and therefore gain the ability to bindligands with bumps. Using "bumped" ligand moieties and receptor moietiesbearing compensatory mutations enhances the specificity and thus thepotency of our reagents. Bumped reagents should not bind to theendogenous, wildtype receptors, which can otherwise act as a "sink"toward transcriptional modulators based on natural ligand moieties.

In preferred transcriptional modulators, the biological activity of theligand is reduced, most preferably lost, by virtue of the linkage to thetranscriptional modulatory portion, or through bump/hole modificationsdescribed herein. For example, as described in the Examples below,derivatives of FK506 can be prepared to yield derivatives that lackimmunosuppressive properties but retain the ability to bind FKBP withhigh affinity (Belshaw, P. J. et al. (1996) Proc. Natl. Acad. Sci. USA93: 4604-4607; Bierer, B. E. et al. (1990) Science 250: 556-559).Similarly, ligands such as cyclosporin A (CsA), which is a cyclicundecapeptide that binds with high affinity (6 nM) to its intracellularreceptor cyclophilin, an 18 kDa monomeric protein, can be modified toreduce or eliminate its immunosuppressive properties. Other ligandswhich can be used are steroids. By way of non-limiting example,glucocorticoids, ecdysone, and estrogens can be used.

Sites of interest for linking of FK506 and analogs thereof includepositions involving annular carbon atoms from about 17 to 24 andsubstituent positions bound to those annular atoms, e.g. 21 (allyl), 22,37, 38, 39 and 40, or 32 (cyclohexyl), while the same positions exceptfor 21 are of interest for FK520. For cyclosporin, sites of interestinclude MeBmt, position 3 and position 8.

Examples of additional modifications include modifying the groups atposition 9 or 10 of FK506 (see Van Duyne et al (1991) Science 252, 839),so as to increase their steric requirement, by replacing the hydroxylwith a group having greater steric requirements, or by modifying thecarbonyl at position 10, replacing the carbonyl with a group havinggreater steric requirements or functionalizing the carbonyl, e.g.forming an N-substituted Schiff's base or imine, to enhance the bulk atthat position. Various functionalities which can be convenientlyintroduced at those sites are alkyl groups to form ethers, acylamidogroups, N-alkylated amines, where a 2-hydroxyethylimine can also form a1,3-oxazoline, or the like. Generally, the substituents will be fromabout 1 to 6, usually 1 to 4, and more usually 1 to 3 carbon atoms, withfrom 1 to 3, usually 1 to 2 heteroatoms, which will usually be oxygen,sulfur, nitrogen, or the like. By using different derivatives of thebasic structure, one can create different ligands with differentconformational requirements for binding. By mutagenizing receptors, onecan have different receptors of substantially the same sequence havingdifferent affinities for modified ligands not differing significantly instructure.

"Bumped" monomeric and dimeric ligands have been prepared using C10acetamide and formamide derivatives of FK506. Spencer et al,"Controlling Signal Transduction with Synthetic Ligands," Science 2625136 (1993): 1019-1024. Preferred "bumped" modifications of derivativesof FK506 include one change at C10 and one at C9 of FK506. The R- andS-isomers of the C10 acetamide and formamide of FK506 have beensynthesized by standard techniques. These bumped derivatives have lostat least three orders of magnitude in their binding affinity towardsFKBP12. The affinities were determined by measuring the ability of thederivatives to inhibit FKBP12's rotamase activity.

An illustrative member of a second class of C9-bumped derivatives is thespiro-epoxide, which has been prepared by adaptation of knownprocedures. See e.g., Fisher et al, J Org Chem 56 8(1991): 2900-7 andEdmunds et al, Tet Lett 32 48 (1991):819-820. A particularly interestingseries of C9 derivatives are characterized by their sp3 hybridizationand reduced oxidation state at C9.

Ligand-Binding Domain

The language "ligand-binding domain" is intended to include a molecule,e.g., a protein, that binds to a selected ligand and initiates aresponse, e.g., a change in transcriptional activity. Preferably, theligand-binding domain is fused to, e.g., covalently linked to, aheterologous moiety to form a chimeric protein. The ligand-bindingdomain can be any convenient moiety which will allow for induction usinga natural or unnatural ligand, preferably an unnatural synthetic ligand.A wide variety of binding proteins, including receptors, are known,which can be used as ligand-binding as indicated above. Of particularinterest are binding proteins for which ligands (preferably smallorganic ligands) are known or may be readily produced. These receptorsor ligand binding moieties include the FKBPs and cyclophilin receptors,the steroid receptors, the tetracycline receptor, the other receptorsindicated above, and the like, as well as "unnatural" receptors, whichcan be obtained from antibodies, particularly the heavy or light chainsubunit, mutated sequences thereof, random amino acid sequences obtainedby stochastic procedures, combinatorial syntheses, and the like. For themost part, the receptor moieties will be at least about 50 amino acids,and fewer than about 350 amino acids, usually fewer than 200 aminoacids, either as the natural moiety or truncated active portion thereof.Preferably the binding moiety will be small (<25 kDa, to allow efficienttransfection in viral vectors), monomeric, nonimmunogenic, and shouldhave synthetically accessible, cell permeant, nontoxic ligands, e.g.,ligand-transcriptional modulating moieties, that can modulatetranscription.

Preferred FKBPs are the cytosolic receptors for macrolides such asFK506, FK520 and rapamycin and are highly conserved across species.Information concerning the nucleotide sequences, cloning, and otheraspects of various FKBP receptors is known in the art, which permittingthe synthesis or cloning of DNA encoding the desired FKBP peptidesequence, e.g., using known methods and PCR primers based on publishedsequences. See e.g. Staendart et al, 1990, Nature 346, 671-674 (humanFKBP12); Kay, 1996, Biochem. J. 314, 361-385 (review). Homologous FKBPproteins in other mammalian species, in yeast, and in other organsimsare also known in the art and may be used in the chimeric proteinsdisclosed herein. See e.g. Kay, 1996, Biochem. J. 314, 361-385 (review).The size of FKBP domains for use in this invention varies, depending onwhich FKBP protein is employed. FKBP may additionally comprise anaturally-occurring peptide sequence derived from the human FKBP12protein or a peptide sequence derived from another human FKBP, from amurine or other mammalian FKBP, or from some other animal, yeast orfungal FKBP. Preferred FKBPs may contain up to about ten (preferably1-5) amino acid substitutions, insertions or deletions within thatregion relative to the naturally-occurring sequence; may be a peptidesequence encoded by a DNA sequence capable of selectively hybridizing toa DNA molecule encoding a naturally-occurring FKBP or may be encoded bya DNA sequence which would be capable, but for the degeneracy of thegenetic code, of selectively hybridizing to a DNA molecule encoding anaturally-occurring FKBP.

"Capable of selectively hybridizing" as that phrase is used herein meansthat two DNA molecules are susceptible to hybridization with oneanother, despite the presence of other DNA molecules, underhybridization conditions which can be chosen or readily determinedempirically by the practitioner of ordinary skill in this art. Suchtreatments include conditions of high stringency such as washingextensively with buffers containing 0.2 to 6×SSC, and/or containing 0.1%to 1% SDS, at temperatures ranging from room temperature to 65-75° C.See for example F. M. Ausubel et al., Eds, Short Protocols in MolecularBiology, Units 6.3 and 6.4 (John Wiley and Sons, New York, 3d Edition,1995).

In certain embodiments, an FKBP peptide sequence for use in chimericproteins will be capable of participating in a dimer, trimer ormultimer, for example, in a complex with a another protein such asFRB-containing protein (as may be determined by any means, direct orindirect, for detecting such binding). "Dimerization","oligomerization"and "multimerization" refer to the association of two or more proteins,mediated, in the practice of this invention, by the binding of each suchprotein to a common ligand. Detailed construction of FKBP,FRB-containing chimeras are described in WO96/41865.

The portion of the construct encoding the receptor can be subjected tomutagenesis for a variety of reasons. The mutagenized protein canprovide for higher binding affinity, allow for discrimination by theligand of the naturally occurring receptor and the mutagenized receptor,provide opportunities to design a receptor-ligand pair, or the like. Thechange in the receptor can involve changes in amino acids known to be atthe binding site, random mutagenesis using combinatorial techniques,where the codons for the amino acids associated with the binding site orother amino acids associated with conformational changes can be subjectto mutagenesis by changing the codon(s) for the particular amino acid,either with known changes or randomly, expressing the resulting proteinsin an appropriate prokaryotic host and then screening the resultingproteins for binding. Detailed protocols for generating such mutationsare provided in WO96/41865. Illustrative of this situation is to modifyFKBP12's Phe36 to Ala and/or Asp37 to Gly or Ala to accommodate asubstituent at positions 9 or 10 of FK506 or FK520. In particular,mutant FKBP12 moieties which contain Val, Ala, Gly, Met or other smallamino acids in place of one or more of Tyr26, Phe36, Asp37, Tyr82 andPhe99 are of particular interest as receptor moieties for FK506-type andFK-520-type ligands containing modifications at C9 and/or C10.

The ability to employ in vitro mutagenesis or combinatorialmodifications of sequences encoding proteins allows for the productionof libraries of proteins which can be screened for binding affinity fordifferent ligands. For example, one can totally randomize a sequence of1 to 5, 10 or more codons, at one or more sites in a DNA sequenceencoding a binding protein, make an expression construct and introducethe expression construct into a unicellular microorganism, and develop alibrary. One can then screen the library for binding affinity to one ordesirably a plurality of ligands. The best affinity sequences which arecompatible with the cells into which they would be introduced can thenbe used as the binding domain. The ligand would be screened with thehost cells to be used to determine the level of binding of the ligand toendogenous proteins. A binding profile could be defined weighting theratio of binding affinity to the mutagenized binding domain with thebinding affinity to endogenous proteins. Those ligands which have thebest binding profile could then be used as the ligand. Phage displaytechniques, as a non-limiting example, can be used in carrying out theforegoing.

Single or multiple mutants of a ligand-binding domain can be generatedby co-randomizing structurally-identified residues that are or would bein contact with or near one or more ligand substituents. For example, acollection of polypeptides containing FKBP domains randomized at theidentified positions can be prepared e.g. using conventional syntheticor genetic methods. Such a collection represents a set of FKBP domainscontaining replacement amino acids at one or more of such positions. Thecollection is screened and FKBP variants are selected which possess thedesired ligand binding properties. In general, randomizing severalresidues simultaneously is expected to yield compensating mutants ofhigher affinity and specificity for a given bumped ligand as itmaximizes the likelihood of beneficial cooperative interactions betweensidechains. Techniques for preparing libraries randomized at discretepositions are known and include primer-directed mutagenesis usingdegenerate oligonucleotides, PCR with degenerate oligonucleotides, andcassette mutagenesis with degenerate oligonucleotides (see for exampleLowman, H. B, and Wells, J. A. Methods: Comp. Methods Enzymol. 1991. 3,205-216; Dennis, M. S. and Lazarus, R. A. 1994. J. Biol. Chem. 269,22129-22136; and references therein).

In many cases, randomization of only the few residues in or near directcontact with a given position in a ligand may not completely explore allthe possible variations in FKBP conformation that could optimallyaccommodate a ligand substituent (bump). Thus the construction is alsoenvisaged of unbiased libraries containing random substitutions that arenot based on structural considerations, to identify subtle mutations orcombinations thereof that confer preferential binding to bumped ligands.Several suitable mutagenesis schemes have been described, includingalanine-scanning mutagenesis (Cunningham and Wells (1989) Science 244,1081-1085), PCR misincorporation mutagenesis (see e.g. Cadwell andJoyce,1992, PCR Meth. Applic. 2, 28-33), and `DNA shuffling` (Stemmer,1994, Nature 370, 389-391 and Crameri et al, 1996, Nature Medicine 2,100-103). These techniques produce libraries of random mutants, or setsof single mutants, that are then searched by screening or selectionapproaches.

In many cases, an effective strategy to identify the best mutants forpreferential binding of a given bump is a combination of structure-basedand unbiased approaches. See Clackson and Wells, 1994, TrendsBiotechnology 12, 173-184 (review). For example we contemplate theconstruction of libraries in which key contact residues are randomizedby PCR with degenerate oligonucleotides, but with amplificationperformed using error-promoting conditions to introduce furthermutations at random sites. A further example is the combination ofcomponent DNA fragments from structure-based and unbiased randomlibraries using DNA shuffling.

A further alternative is to clone the randomized ligand-binding domainsequences into a vector for phage display, allowing in vitro selectionof the variants that bind best to the ligand. Affinity selection invitro may be performed in a number of ways. For example, rapalog ismixed with the library phage pool in solution in the presence ofrecombinant receptor tagged with an affinity handle (for example ahexa-histidine tag, or GST), and the resultant complexes are captured onthe appropriate affinity matrix to enrich for phage displaying receptorharboring complementary mutations. Techniques for phage display havebeen described, and other in vitro selection selection systems can alsobe contemplated (for example display on lambda phage, display onplasmids, display on baculovirus). Furthermore, selection and screeningstrategies can also be used to improve other properties of benefit inthe application of this invention, such as enhanced stability in vivo.For a review see Clackson, T. and Wells, J. A. 1994. Trends Biotechnol.12, 173-184.

Antibody subunits, e.g. heavy or light chain, particularly fragments,more particularly all or part of the variable region, or fusions ofheavy and light chain to create high-affinity binding, can be used asthe binding moiety. Antibodies can be prepared against haptenicmolecules which are physiologically acceptable and the individualantibody subunits screened for binding affinity. The cDNA encoding thesubunits can be isolated and modified by deletion of the constantregion, portions of the variable region, mutagenesis of the variableregion, or the like, to obtain a binding protein moiety that has theappropriate affinity for the ligand. In this way, almost anyphysiologically acceptable haptenic compound can be employed as theligand or to provide an epitope for the ligand. Instead of antibodyunits, natural receptors can be employed, where the binding moiety isknown and there is a useful ligand for binding.

Transcriptional Modulating Portions (TMP)

The transcriptional modulators of the invention additionally include atleast one transcriptional modulating portion (TMP). The language"transcriptional modulating portion" is intended to include a portionwhich modulates transcription including chemical moieties andproteinaceous domains. The preferred TMPs of the present invention arechemical moieties, e.g., non-peptidyl, small molecules (e.g., having amolecular weight of less than about 1000).

The terms "chemical moiety" or "moiety" are intended to includesynthetic and naturally-occurring non-proteinaceous entities. Forexample, chemical moieties include unsubstituted or substituted alkyl,aromatic, or heterocyclyl moieties including macrolides, leptomycins andrelated natural products or analogs thereof.

The terms "proteinaceous domain" is intended to includenaturally-occurring and nonnaturally-occurring polypeptides. The termsprotein, peptide, and polypeptide are used interchangeably herein.

Preferably, the TMP is small having a molecular weight of less thanabout 5 kD, less than about 4 kD, preferably less than 3 kD, and evenmore preferably, less than 1.5 kD. When the TMP is a peptide, its aminoacid sequence can range in size from about 5 to 30 amino acids, morepreferably from about 10 to 25 amino acids, and most preferably fromabout 15 to 20 amino acids. The peptide can be of a size within a rangeusing any of the above-recited numbers as the upper or lower value ofthe range.

The transcriptional modulators of the present invention are capable ofmodulating, e.g., stimulating or inhibiting, transcription of a gene,e.g., a target gene. As used herein, the terms "modulation oftranscription" or "regulation of gene expression" or variations thereofare intended to include any changes in gene expression which aretriggered directly or indirectly by the transcriptional modulators ofthe invention, preferably by the transcriptional modulating portion ofthese transcriptional modulators. For example, changes in geneexpression can occur as a result of one or more of: (i) a direct orindirect interaction(s) of the transcriptional modulator with acomponent of the transcriptional machinery; (ii) an alteration(s) ofchromatin structure; and (iii) an alteration(s) in the effectiveconcentration of the transcriptional modulator in the nucleus of a cell.

An example of a transcriptional modulator that regulates gene expressionvia an interaction with a component of the transcriptional machinery isillustrated in schematic form in FIG. 1C. As indicated, thetranscriptional modulator is depicted between a DNA-bound chimericprotein (illustrated as GAL4-FKBP) and the basal transcriptionalmachinery which is bound to the core promoter region. The high affinityinteraction between the ligand-binding domain of the chimeric protein(the FKBP illustrated in FIG. 1C) and the transcriptional modulatorbrings the transcriptional modulating portion in close proximity withthe target gene, thus triggering changes in gene expression. Thetranscriptional modulating portion can include a transcription activatorportion, which recruits and/or activates the basal transcriptionmachinery directly or indirectly to result in activation or enhancementof gene expression. Alternatively, the transcriptional modulator portionmay include a transcription repressor portion which inhibits ordecreases gene expression. Assays for identifying transcriptionalmodulators which activate or repress transcription activity can beperformed using procedures known in the art as exemplified by theExamples provided herein.

An example of a transcriptional modulator that regulates gene expressionby altering the chromatin structure occurs when the transcriptionalmodulator, particularly its transcriptional modulating portion,interacts with chromatin-remodeling and/or modifying complex. In suchcases, addition of the transcriptional modulator results in remodelingof the chromatin assembly to, e.g., facilitate assembly of transcriptioncomplexes. For example, transcriptional coactivators having intrinsichistone acetyl transferase activity (HATs) have been reported tointeract with target histones bound to DNA and overcome the inhibitoryeffect of chromatin on gene expression (Jenster, G. et al. (1997) Proc.Natl. Acad. Sci. USA 94(15): 7879-84). In those instances wheretranscription activation is desired, the high affinity interactionbetween the ligand-binding domain of the chimeric protein and thetranscriptional modulator can be used to bring the chromatin-remodelingand/or modifying complex in close proximity to the target gene, thusactivating gene expression. Alternatively, transcriptional activity maybe inhibited by a transcriptional modulator that recruits to the targetgene a chromatin-modifying component such that as histone deacetylasesuch that transcriptional activity is repressed. Assays for detectingchanges in chromatin structure and alterations in HAT activity are knownin the art. See e.g., Spencer, T. E. et al. (1997) Nature 389: 194-8;Jenster, G. et al. (1997) supra; Steger, D. J. et al. (1997) Methods 12(3): 276-85; Steger, D. J. et al. (1996) 18(11)).

An example of a transcriptional modulator that regulates gene expressionby altering the effective concentration of a complex of atranscriptional modulator and a chimeric protein in the cell nucleus isprovided when the transcriptional modulator, preferably, thetranscriptional modulating portion, is capable of interacting with acomponent of the nuclear pore, e.g., a nuclear importer or an exporter,such that the interaction between the transcriptional modulator and thechimeric protein results in translocation of the transcriptionalmodulator-chimeric protein complex through the nuclear pore. Forexample, the transcriptional modulator can include a nuclear import or anuclear export signal, or alternatively a chemical moiety whichinteracts with a component of the nuclear pore, e.g., e.g., a chemicalmoiety that interacts with a nuclear exporter, e.g., a member of theleptomycin and kazusamycin family of antibiotics. In such examples, thechimeric protein preferably includes at least one DNA-binding domain, aligand-binding domain and a transcription modulating domain.

In those embodiments where an increase in the effective concentration ofthe complex of the transcriptional modulator and the chimeric protein isdesired, a chimeric protein is constructed such that it remains in thecytoplasm of a cell. The cell is contacted with a transcriptionalmodulator which includes, e.g., a nuclear import signal, and thus thecomplex of the chimeric protein and transcriptional modulator istranslocated into the nucleus. Such translocation increases theeffective concentration of the chimeric protein in the nucleus. Iftranscriptional activation is desired, the chimeric protein isconstructed such that it contains a transcriptional activation domain.Alternatively, if transcriptional repression is desired, the chimericprotein should contain a transcriptional repressor domain.

In other embodiments, modulation of gene expression can result from adecrease in the effective concentration of a transcriptional modulator.For example, a chimeric protein containing at least one DNA-bindingdomain, a ligand-binding domain and a transcription modulating domaincan be prepared such that it is present in the nucleus. Thetranscriptional modulating domain can include a transcriptionalactivating domain, thus resulting in constitutive activation of geneexpression. In other embodiments, the transcriptional modulating domaincan include a transcriptional repressor domain, thus repressing geneexpression. Upon contacting the cell with a transcriptional modulatorwhich includes a nuclear export signal, the complex of the chimericprotein and transcriptional modulator is translocated outside thenucleus. Such translocation decreases the effective concentration of thechimeric protein in the nucleus. Thus, transcriptional activity will bereduced in those instances where the chimeric protein contains atranscriptional activating domain. Alternatively, transcriptionalactivity will be increased in those instances where the chimeric proteincontains a transcriptional repressor domain. Assays for detectingchanges nuclear import-export activity are known in the art. See e.g.,Wolff, B. (1997) Chemistry and Biology, Vol. 4(2); for a description ofan in vitro system using semi-permeabilized cells and a fluorescentimport substrate see Paschal, B. M. and Gerace, L. (1995) J. Cell Biol.129: 925-937.

As described above, transcriptional modulators can include chemicalmoieties and proteinaceous domains. The TMPs (including chemicalmoieties) also can be designed to form compounds which modulatetranscription, e.g., by modelling the TMPs after known transcriptionalactivators or repressors, nuclear importers or nuclear exporters. In oneembodiment, TMPs (in unlinked forms) are screened for a desiredactivity, e.g., binding (e.g., nuclear export and import), ortranscriptional modulation activity. In this embodiment, the TMP is notlinked to the selected ligand until after it is identified as a TMP ofthe present invention.

Exemplary chemical moieties are those identified using the methodsprovided herein. An additional example of a TMP of the invention whichis a chemical moiety is a member of the leptomycin and kazusamycinfamily of antibiotics. These antibiotics have been shown to bind withhigh affinity to a component of the nuclear export (Wolff, B. (1997)supra). Members of this family of antibiotics have the general formulaas follows: ##STR1## wherein R₁ and R₂ are each independently selectedfrom a group consisting of a hydrogen, a lower alkyl, an alkyl, ahydroxyalkyl, and a haloalkyl. The term alkyl as herein is intended toinclude both "unsubstituted alkyls" and "substituted alkyls", the latterof which refers to alkyl moieties having substituents replacing ahydrogen on one or more carbons of the hydrocarbon backbone. Suchsubstituents can include, for example, halogen (including fluoroalkyl),hydroxyl (including hydroxyalkyl). Unless the number of carbons isotherwise specified, "lower alkyl" as used herein means an alkyl group,as defined above, but having from one to ten carbons, more preferablyfrom one to six, and most preferably from one to four carbon atoms inits backbone structure, which may be straight or branched-chain.Examples of lower alkyl groups include methyl, ethyl, n-propyl,i-propyl, tert-butyl, hexyl, heptyl, octyl and so forth. In preferredembodiment, the term "lower alkyl" includes a straight chain alkylhaving 2 or fewer carbon atoms in its backbone. As used herein, the term"halogen" designates --F, --Cl, --Br or --I; the term "sulfhydryl" or"thiol" means --SH; the term "hydroxyl" means --OH. Preferredsubstitutions of R₁ include --CH₃ or CH₂ OH; preferred R₂ substitutionsinclude CH₃, or CH₂ CH₃. The TMP can be a member of this family ofantibiotics or a structural variant thereof which retains its affinityto bind with a component of the nuclear export.

TMPs can include both naturally occurring and synthetic compounds. Incertain embodiments, the TMP is a peptide. The peptide can include oneor more amino acids, and preferably all amino acids having either the L-or the D- stereochemistry. Peptides including any combination of aminoacids having the L- or D- stereochemistry are encompassed by theinvention. Preferred configurations are those which render a peptidemore resistant to proteolytic cleavage, as described by theD-configuration of amino acid sequences 1-29 of VP 16 provided in theExamples herein. Peptides can be chemically synthesized using techniquesknown in the art such as conventional Merrifield solid phase f-Moc ort-Boc chemistry. Alternatively, the peptides can be producedrecombinantly by standard techniques. Peptide mimetics can also begenerated which show enhanced stability. For instance, non-hydrolyzablepeptide analogs can be generated using benzodiazepine (e.g., seeFreidinger et al. in Peptides: Chemistry and Biology, G. R. Marshalled., ESCOM Publisher: Leiden, Netherlands, 1988), azepine (e.g., seeHuffman et al. in Peptides: Chemistry and Biology, G. R. Marshall ed.,ESCOM Publisher: Leiden, Netherlands, 1988), substituted gama lactamrings (Garvey et al. in Peptides: Chemistry and Biology, G. R. Marshalled., ESCOM Publisher: Leiden, Netherlands, 1988), keto-methylenepseudopeptides (Ewenson et al. (1986) J Med Chem 29:295; and Ewenson etal. in Peptides: Structure and Function (Proceedings of the 9th AmericanPeptide Symposium) Pierce Chemical Co. Rockland, Ill., 1985), b-turndipeptide cores (Nagai et al. (1985) Tetrahedron Lett 26:647; and Satoet al. (1986) J Chem Soc Perkin Trans 1:1231), and b-aminoalcohols(Gordon et al. (1985) Biochem Biophys Res Commun 126:419; and Dann etal. (1986) Biochem Biophys Res Commun 134:71).

In some embodiments, the TMP includes a portion of a transcriptionactivator. The transcription activators can be endogenous or exogenousto the cellular host. If the transcription factors are exogenous, butfunctional within the host and can cooperate with the endogenous RNApolymerase (rather than requiring an exogenous RNA polymerase, for whicha gene could be introduced), then an exogenous promoter elementfunctional with the fused transcription factors can be provided with asecond construct for regulating transcription of the target gene. Bythis means the initiation of transcription can be restricted to thegene(s) associated with the exogenous promoter region, i.e., the targetgene(s).

Transcriptional activation domains found within various proteins havebeen grouped into categories based upon similar structural features.Types of transcription activation domains which can be used as TMPs ofthe present invention include acidic transcription activation domains,proline-rich transcription activation domains, serine/threonine-richtranscription activation domains and glutamine-rich transcriptionactivation domains, and/ or fragments thereof. Examples of acidictranscriptional activation domains include the VP16 amino acid residues413-490, residues 753-881 of GAL-4. Examples of proline-rich activationdomains include amino acids 399-499 of CTF/NF1 and amino acid residues31-76 of AP2. Examples of serine/threonine-rich transcription activationdomains include residues 1-427 of ITF1. Examples of glutamine-richactivation domains include amino acid residues 175-269 of Oct1 and aminoacid residues132-243 of Sp1. The amino acid sequences of each of theabove-described regions are disclosed in Seipel, K. et al. (1992) EMBOJ. 13: 4961-4968.

As an alternative to using a naturally-occurring activation domains ormodified form thereof, the TMP can be one of the binding proteinsassociated with bridging between a transcriptional activation domain andan RNA polymerase, including but not limited to RNA polymerase II. Theseproteins include the proteins referred to as TAF's, the TFII proteins,e.g., TFIIB and TFIID, or the like. Thus, one can use any one orcombination of proteins, for example, fused proteins or binding motifsthereof, which serve in the bridge between the DNA binding protein andRNA polymerase and provide for initiation of transcription.

Rather than having a transcriptional activation domain as the TMP, aninactivation domain, such as ssn-6/TUP-1 or Kruppel-family suppressordomain, can be employed as the TMP. In this manner, regulation resultsin turning off the transcription of a gene which is constitutivelyexpressed. For example, in the case of gene therapy one can provide forconstitutive expression of a hormone, such as growth hormone, bloodproteins, immunoglobulins, etc. By employing transcriptional repressorsas the TMP(s), e.g., ligands covalently linked to an inactivationmoiety, the expression of the gene can be inhibited.

Peptide sequences containing nuclear import or export signals can beused ias TMPs in those embodiments where nuclear import or exportactivity is desired. For example, nuclear import or nuclear localizationsequence are known in the art and are known to have a plurality of basicamino acids, referred to as a bipartite basic repeat (reviewed inGarcia-Bustos et al., Biochimica et Biophysica Acta (1991) 1071, 83-101;Imamoto, N.). Examples of nuclear export signals (NES) includeleucine-rich amino acid peptide sequences as described in CRM1 proteinand various viral proteins such as HIV-1 Rev protein, and E1B and E4proteins (Ossareh-Nazari, B. et al. (1997) Science 278: 141-4; Wolff, B.(1997) supra; Dobelstein, M. (1997) EMBO J. 16(4): 4276-84).

Linkers

The linkage between the ligand and the TMP is selected such that thelinkage between the (at least two) components of the transcriptionalmodulator of this invention occurs, and the transcriptional modulatorperforms its intended function(s). The linker is preferably covalent,either a covalent bond or a linker moiety covalently attached to theligand moiety and TMP. In certain embodiments, the linker of theinvention can a non-covalent bond, e.g., an organometallic bond througha metal center such as platinum atom. For covalent linkages, variousfunctionalities can be used, such as amide groups, including carbonicacid derivatives, ethers, esters, including organic and inorganicesters, amino, urethane, urea, or the like. To provide for linking, theparticular ligand monomer can be modified by oxidation, hydroxylation,substitution, reduction, etc., to provide a site for coupling. Dependingon the monomer, various sites can be selected as the site of coupling.It will be appreciated that modifications which do not significantlydecrease the ability of the ligand to bind to its receptor arepreferred.

The ligands can be synthesized by any convenient means, where thelinking group will be at a site which allows the formed transcriptionalmodulator to perform its intended function, e.g., does not interferewith the binding of the binding site of a ligand to the ligand-bindingprotein, or with the activity of the transcriptional modulating portion.For example, the linker group can be attached to a FK506 derivative,bearing a hydroxyethyl group at C-21, and further attach to an aminoacid of a transcriptional modulating portion. Where the active site forphysiological activity and binding site of a ligand to theligand-binding protein are different, it will usually be desirable tolink at the active site to inactivate the ligand.

Various linking groups can be employed, usually of from 1-30, moreusually from about 1-20 atoms (other than hydrogen) in the chain betweenthe two moieties, where the linking groups will be primarily composed ofcarbon, hydrogen, nitrogen, oxygen, sulfur and phosphorous. Preferably,the linker is an achiral linker. The linking groups can involve a widevariety of functionalities, such as amides and esters, both organic andinorganic, amines, ethers, thioethers, disulfides, quaternary ammoniumsalts, hydrazines, etc. The chain can include aliphatic, alicyclic,aromatic or heterocyclic groups. The chain will be selected based onease of synthesis and the stability of the ligand-transcriptionalmodulating moiety. Thus, if one wishes to maintain long-term activity, arelatively inert chain will be used, so that the ligand-transcriptionalmodulating moiety link will not be cleaved. Alternatively, if one wishesonly a short half-life in the blood stream, then various groups can beemployed which are readily cleaved, such as esters and amides,particularly peptides, where circulating and/or intracellular proteasescan cleave the linking group.

Various groups can be employed as the linking group between ligands,such as alkylene, usually of from 2 to 20 carbon atoms, azalkylene(where the nitrogen will usually be between two carbon atoms), usuallyof from 4 to 18 carbon atoms), N-alkylene azalkylene (see above),usually of from 6 to 24 carbon atoms, arylene, usually of from 6 to 18carbon atoms, ardialkylene, usually of from 8 to 24 carbon atoms,bis-carboxamido alkylene of from about 8 to 36 carbon atoms, etc.Illustrative groups include decylene, octadecylene, 3-azapentylene,5-azadecylene, N-butylene 5-azanonylene, phenylene, xylylene,p-dipropylenebenzene, bis-benzoyl 1,8-diaminooctane and the like.

Chimeric Proteins

"Recombinant" or "chimeric" proteins, as those terms are used herein,indicate proteins having two or more heterologous domains or portions,e.g., polypeptide domains or sequences which are mutually heterologousin the sense that they do not occur together in the same arrangement innature. More specifically, the component portions are not found in thesame continuous polypeptide or nucleotide sequence or molecule innature, at least not in the same order or orientation or with the samespacing present in the chimeric protein or recombinant DNA molecule ofthis invention.

Preferably, the chimeric proteins of the invention have a ligand-bindingdomain which is capable of binding to a selected ligand molecule and aDNA-binding domain which is capable of binding to a particular DNAsequence(s). The DNA-binding domain may be naturally-occurring or not,including recombinant DNA-binding domain. The chimeric protein may alsoinclude one or more linker regions comprising one or more amino acidresidues, or include no linker, as appropriate, to join the selecteddomains.

The chimeric proteins may contain additional domains. For example,chimeric proteins used in the nuclear translocation mechanism forcontrolling transcriptional regulation described above may additionallycontain a transcriptional modulatory domain, e.g., a transcriptionalactivation or repressor domain.

Such chimeric proteins and DNA sequences which encode them arerecombinant in the sense that they contain at least two constituentportions which are not otherwise found directly linked (covalently)together in nature, at least not in the order, orientation orarrangement present in the recombinant material. Desirable properties ofthe chimeric proteins of the invention include high affinity forspecific nucleotide sequences, low affinity for most other sequences ina complex genome (such as a mammalian genome), and low dissociationrates from specific DNA sites. Preferably, the DNA-binding domains bindto a particular DNA sequence(s) with high affinity, preferably with adissociation constant below about 10⁻⁹ M, more preferably below about10⁻¹⁰ M, even more preferably below 10⁻¹¹ M.

The choice of DNA-binding domains may be influenced by a number ofconsiderations, including the species, system and cell type which istargeted; the feasibility of incorporation into a chimeric protein, asmay be shown by modeling; and the desired application or utility. Thechoice of DNA-binding domains may also be influenced by the individualDNA sequence specificity of the domain and the ability of the domain tointeract with other proteins or to be influenced by a particularcellular regulatory pathway. Preferably, the distance between domaintermini is relatively short to facilitate use of the shortest possiblelinker or no linker. The DNA-binding domains can be isolated from anaturally-occurring protein, or may be a synthetic molecule based inwhole or in part on a naturally-occurring domain.

As used herein, a "DNA-binding domain" refers to a molecule, e.g., aprotein, which binds to a specific DNA sequence(s). The DNA bindingdomain of the chimeric protein may be derived from any vertebrate,nonvertebrate, fungal, plant, or bacterial source including but notlimited to GAL4 (Keegan et al. (1986) Science 231: 699-704), ADR1(Hartshorne et al. (1986) Nature 320: 283-287), SwI (Stillman et al.(1988) EMBO J. 7: 485-495) and as generally reviewed in Johnson et al.(1989) Ann. Rev. Biochem. 58: 799-839). It may be a repressor proteinsuch as, for example, the Lex A. DNA-binding domains found in variousproteins have been grouped into categories based upon similar structuralfeaturs. Such types of DNA binding domains are recognized in the art,such as zinc fingers (Miller et al. (1985) EMBO J. 4: 1609),homeodomains (Scott et al., Biochim. Biophys. Acta 989:25-48 (1989) andRosenfeld, Genes Dev. 5:897-907 (1991)).

Chimeric constructs encoding chimeric proteins can optionally containcellular targeting sequences, e.g., signal consensus sequence, whichprovide for the protein to be translocated to the nucleus. As describedabove, this "signal consensus" sequence has a plurality of basic aminoacids, referred to as a bipartite basic repeat (reviewed inGarcia-Bustos et al., Biochimica et Biophysica Acta (1991)1071, 83-101).This sequence can appear in any portion of the molecule internal orproximal to the N- or C-terminus and results in the chimeric proteinbeing transported inside the nucleus. However, in those embodiments inwhich transcriptional activity is regulated by nuclear translocation,i.e., nuclear import, the chimeric protein will not contain such signalconsensus sequence.

Nucleotide sequences encoding chimeric proteins can be placed under thecontrol of a suitable promoter sequence. It may be desirable for thenucleotide sequences encoding chimeric protein to be placed under thecontrol of a constitutively active promoter sequence, although thechimeric protein may also be placed under the control of an induciblepromoter, such as the metallothionine promoter (Brinster et al., 1982,Nature 296:39-42) or a tissue specific promoter. Promoter sequenceswhich may be used according to the invention include, but are notlimited to, the SV40 early promoter region (Bernoist and Chambon, 1981,Nature 290:304-310), the promoter contained in the long terminal repeatof Rous sarcoma virus (Yamamoto, et al., 1980, Cell 22:787-797), theherpes thymidine kinase promoter (Wagner et al., 1981, Proc. Natl. Acad.Sci. U.S.A. 78:144-1445) the human cytomegalovirus (CMV) immediate earlypromoter/enhancer (Boshart et al., 1985, Cell 41:521-530).

It will be preferred in certain embodiments, that the chimeric proteinsbe expressed in a cell-specific or tissue-specific manner. Suchspecificity of expression may be achieved by operably linking one oremore of the DNA sequences encoding the chimeric protein(s) to acell-type specific transcriptional regulatory sequence (e.g.promoter/enhancer). Numerous cell-type specific transcriptionalregulatory sequences are known. Others may be obtained from genes whichare expressed in a cell-specific manner. See e.g. PCT/US95/10591,especially pp. 36-37.

For example, constructs for expressing the chimeric proteins may containregulatory sequences derived from known genes for specific expression inselected tissues. Representative examples are tabulated below:

    __________________________________________________________________________    Tissue                                                                              Gene     Reference                                                      __________________________________________________________________________    lens  γ2-crystallin                                                                    Breitman, M.L., Clapoff, S., Rossant, J., Tsui, L.C.,                            Golde, L.M., Maxwell, I.H., Bernstin, A. (1987)                               Genetic Ablation: targeted expression of a toxin gene                         causes microphthalmia in transgenic mice. Science                             238: 1563-1565                                                 αA- Landel, C.P., Zhao, J., Bok, D., Evans, G.A. (1988)                 crystallin Lens-specific expression of a recombinant ricin                     induces developmental defects in the eyes of                                  transgenic mice. Genes Dev. 2: 1168-1178                                      Kaur, S., key, B., Stock, J., McNeish, J.D., Akeson,                          R., Potter, S.S. (1989) Targeted ablation of alpha-                           crystallin-synthesizing cells produces lens-deficient                         eyes in transgenic mice. Development 105: 613-619                           pituitary Growth Behringer, R.R., Mathews, L.S., Palmiter, R.D.,                            -- hormone Brinster, R.L. (1988) Dwarf mice produced by                       somatrop  genetic ablation of growth hormone-expressing                      cells.                                                           hic cells  Genes Dev. 2: 453-461                                              pancreas Insulin- Ornitz, D.M., Palmiter, R.D., Hammer, R.E., Brinster,        Elastase- R.L., Swift, G.H., MacDonald, R.J. (1985) Specific                  acinar cell expression of an elastase-human growth fusion in                  specific pancreatic acinar cells of transgeneic mice. Nature                   131: 600-603                                                                  Palmiter, R.D., Behringer, R.R., Quaife, C.J.,                                Maxwell, F., Maxwell, I.H., Brinster, R.L. (1987)                             Cell lineage ablation in transgeneic mice by cell-                            specific expression of a toxin gene. Cell 50: 435-443                       T cells lck promoter Chaffin, K.E., Beals, C.R., Wilkie, T.M., Forbush,         K.A., Simon, M.I., Perlmutter, R.M. (1990) EMBO                               Journal 9: 3821-3829                                                        B cells Immunoglobulin Borelli, E., Heyman, R., Hsi, M., Evans, R.M.                       (1988)                                                            kappa Targeting of an inducible toxic phenotype in animal                     light chain cells. Proc. Natl. Acad. Sci. USA 85: 7572-7576                    Heyman, R.A., Borrelli, E., Lesley, J., Anderson, D.,                         Richmond, D.D., Baird, S.M., Hyman, R., Evans,                                R.M. (1989) Thymidine kinase obliteration: creation                           of transgenic mice with controlled                                            immunodeficiencies. Proc. Natl. Acad. Sci. USA 86:                            2698-2702                                                                   Schwann P.sub.O promoter Messing, A., Behringer, R R, Hammang, J.P.                         cells  Palmiter, RD, Brinster, RL, Lemke, G., P.sub.0                        promoter                                                           directs espression of reporter and toxin genes to                             Schwann cells of transgenic mice. Neuron 8: 507-520                           1992                                                                         Myelin basic Miskimins, R. Knapp, L., Dewey, MJ, Zhang, X. Cell                             protein and tissue-specific expression of a heterologous                    gene                                                               under control of the myelin basic protein gene                                promoter in trangenic mice. Brain Res Dev Brain Res                           1992 Vol 65: 217-21                                                         spermatids protamine Breitman, M.L., Rombola, H., Maxwell, I.H.,                              Klintworth, G.K., Bernstein, A. (1990) Genetic                  ablation in transgenic mice with attenuated diphtheria                        toxin A gene. Mol. Cell. Biol. 10: 474-479                                  lung Lung Ornitz, D.M., Palmiter, R.D., Hammer, R.E., Brinster,                              surfacant R.L., Swift, G.H., MacDonald, R.J. (1985)                         Specific                                                          gene expression of an elastase-human growth fusion in                          pancreatic acinar cells of transgeneic mice. Nature                           131: 600-603                                                                adipocyte  Ross, S.R, Braves, RA, Spiegelman, BM Targeted                     P2  expression of a toxin gene to adipose tissue:                               transgenic mice resistant to obesity Genes and Dev 7:                         1318-24 1993                                                                muscle myosin light Lee, KJ, Ross, RS, Rockman, HA, Harris, AN,                              chain O'Brien, TX, van-Bilsen, M., Shubeita, HE,                            Kandolf,                                                           R., Brem, G., Prices et al J. Biol. Chem. 1992 Aug 5,                         267: 15875-85                                                                Alpha actin Muscat, GE., Perry, S., Prentice, H. Kedes, L. The                               human skeletal alpha-actin gene is regulated by a                             muscle-specific enhancer that binds three nuclear                             factors. Gene Expression 2, 111-26, 1992                      neurons neurofilament Reeben, M. Halmekyto, M. Alhonen, L. Sinervirta,                     R.                                                                proteins Saarma, M. Janne, J. Tissue-specific expression of rat                              light neurofilament promoter-driven reporter gene in                          transgenic mice. BBRC 1993: 192: 465-70                       liver tyrosine                                                                 aminotransferase                                                              albumin,                                                                      apolipoproteins                                                            __________________________________________________________________________

Additional examples of tissue-specific regulatory sequences includeElastase I gene control region which is active in pancreatic acinarcells (Swift et al., 1984, Cell 38:639-646; Ornitz et al., 1986, ColdSpring Harbor Symp. Quant. Biol. 50:399-409; MacDonald, 1987, Hepatology7:425-515); insulin gene control region which is active in pancreaticbeta cells (Hanahan, 1985, Nature 315:115-122), immunoglobulin genecontrol region which is active in lymphoid cells (Grosschedl et al.,1984, Cell 38:647-658; Adames et al., 1985, Nature 318:533-538;Alexander et al., 1987, Mol. Cell. Biol. 7:1436-1444), mouse mammarytumor virus control region which is active in testicular, breast,lymphoid and mast cells (Leder et al., 1986, Cell 45:485-495), albumingene control region which is active in liver (Pinker et al. 1987, Genesand Devel. 1:268-276), alpha-fetoprotein gene control region which isactive in liver (Krumlauf et al., 1985, Mol. Cell. Biol. 5:1639-1648;Hammer et al., 1987, Science 235:53-58); alpha 1-antitrypsin genecontrol region which is active in the liver (Kelsey et al, 1987, Genesand Devel. 1:161-171), beta-globin gene control region which is activein erythroid cells (Mogram et al., 1985, Nature 315:338-340; Kollias etal., 1986, Cell 46:89-94); myelin basic protein gene control regionwhich is active in oligodendrocyte cells in the brain (Readhead et al.,1987, Cell 48:703-712); myosin light chain-2 gene control region whichis active in skeletal muscle (Sani, 1985, Nature 314:283-286), andgonadotropic releasing hormone gene control region which is active inhypothalamus (Mason et al., 1986, Science 234:1372-1378).

A detailed description of DNA constructs encoding these chimericproteins (and accessory constructs such as DNA constructs encodingtarget genes) is provided in the section entitled "Genes and Vectors"below.

Genes and Vectors

Recombinant nucleic acid molecules containing target genes andnucleotide sequences encoding the chimeric proteins are provided, as arevectors capable of directing their expression, particularly ineukaryotic cells, of which yeast and animal cells are of particularinterest. In view of the constituent components of the chimericproteins, the recombinant nucleic acid molecules which encode them arecapable of selectively hybridizing (a) to a DNA molecule encoding agiven chimeric protein's ligand-binding domain (e.g., FRB domain or FKBPdomain) or a protein containing such a domain and (b) to a DNA moleculeencoding the heterologous domain or a protein from which theheterologous protein domain was derived, e.g., a DNA-binding domain.DNAs are also encompassed which would be capable of so hybridizing butfor the degeneracy of the genetic code.

In the present specification, the terms "plasmid","vector" or"construct" are used interchangeably. As used herein, these terms referto a nucleic acid molecule capable of transporting another nucleic acidto which it has been linked. One type of preferred vector is an episome,i.e., a nucleic acid capable of extra-chromosomal replication. Preferredvectors are those capable of autonomous replication and/expression ofnucleic acids to which they are linked. Vectors capable of directing theexpression of genes to which they are operatively linked are referred toherein as "expression vectors". In general, expression vectors ofutility in recombinant DNA techniques are often in the form of"plasmids" which refer to circular double stranded DNA loops which, intheir vector form are not bound to the chromosome. However, theinvention is intended to include such other forms of expression vectorswhich serve equivalent functions and which become known in the artsubsequently hereto.

The constructs of the invention can be introduced as one or more DNAmolecules or constructs, where there will usually be at least one markerand there may be two or more markers, which will allow for selection ofhost cells which contain the construct(s). The constructs can beprepared in conventional ways, where the genes and modulatory regionsmay be isolated, as appropriate, ligated, cloned in an appropriatecloning host, analyzed by restriction or sequencing, or other convenientmeans. Particularly, using PCR, individual fragments including all orportions of a functional unit may be isolated, where one or moremutations may be introduced using "primer repair",ligation, in vitromutagensis, etc. as appropriate. The construct(s) once completed anddemonstrated to have the appropriate sequences may then be introducedinto the host cell by any convenient means. The constructs may beintegrated and packaged into non-replicating, defective viral genomeslike Adenovirus, Adeno-associated virus (AAV), or Herpes simplex virus(HSV) or others, including retroviral vectors, for infection ortransduction into cells. The constructs may include viral sequences fortransfection, if desired. Alternatively, the construct may be introducedby fusion, electroporation, biolistics, transfection, lipofection, orthe like. The host cells will usually be grown and expanded in culturebefore introduction of the construct(s), followed by the appropriatetreatment for introduction of the construct(s) and integration of theconstruct(s). The cells will then be expanded and screened by virtue ofa marker present in the construct. Various markers which may be usedsuccessfully include hprt, neomycin resistance, thymidine kinase,hygromycin resistance, etc.

In some instances, one may have a target site for homologousrecombination, where it is desired that a construct be integrated at aparticular locus. For example, can knock-out an endogenous gene andreplace it (at the same locus or elswhere) with the gene encoded for bythe construct using materials and methods as are known in the art forhomologous recombination. Alternatively, instead of providing a gene,one may modify the transcriptional initiation region of an endogenousgene to be responsive to the signal initiating moiety. In suchembodiments, transcription of an endogenous gene such as EPO, tPA, SOD,or the like, would be controlled by administration of the ligand. Forhomologous recombination, a number of vectors can be used. See, forexample, Thomas and Capecchi, Cell (1987) 51, 503-512; Mansour, et al.,Nature (1988) 336, 348-352; and Joyner, et al., Nature (1989) 338,153-156.

The constructs may be introduced as a single DNA molecule encoding allof the genes, or different DNA molecules having one or more genes. Theconstructs may be introduced simultaneously or consecutively, each withthe same or different markers.

Vectors containing useful elements such as bacterial or yeast origins ofreplication, selectable and/or amplifiable markers, promoter/enhancerelements for expression in procaryotes or eucaryotes, etc. which may beused to prepare stocks of construct DNAs and for carrying outtransfections are known in the art, and many are commercially available.

IV. Pharmaceutical Compositions

In another aspect, the present invention provides pharmaceuticallyacceptable compositions which comprise a therapeutically-effectiveamount of one or more of the transcriptional modulators of the presentinvention, formulated together with one or more pharmaceuticallyacceptable carrier(s). The pharmaceutical compositions and methodsdescribed herein can include one or more transcriptional modulators ofthe present invention. Any combination of these transcriptionalmodulators (e.g., transcriptional modulators which result in activationof gene expression by any of the mechanisms (i)-(iii)) is intended to beencompassed by the present invention.

The phrase "therapeutically-effective amount" as used herein means thatamount of a transcriptional modulators, or composition comprising such acompound which is effective for the transcriptional modulator to produceits intended function, e.g., the modulation of gene expression. Theeffective amount can vary depending on such factors as the type of cellgrowth being treated or inhibited, the particular type oftranscriptional modulator, the size of the subject, or the severity ofthe undesirable cell growth or activity. One of ordinary skill in theart would be able to study the aforementioned factors and make thedetermination regarding the effective amount of the transcriptionalmodulator without undue experimentation.

The phrase "pharmaceutically acceptable" is employed herein to refer tothose transcriptional modulators, compositions containing suchcompounds, and/or dosage forms which are, within the scope of soundmedical judgment, suitable for use in contact with the tissues of humanbeings and animals without excessive toxicity, irritation, allergicresponse, or other problem or complication, commensurate with areasonable benefit/risk ratio.

Transcriptional modulators of the present invention can exist in freeform or, where appropriate, in salt form. Pharmaceutically acceptablesalts and their preparation are well-known to those of skill in the art.The pharmaceutically acceptable salts of such compounds include theconventional non-toxic salts or the quaternary ammonium salts of suchcompounds which are formed, for example, from inorganic or organic acidsof bases.

The compounds of the invention may form hydrates or solvates. It isknown to those of skill in the art that charged compounds form hydratedspecies when lyophilized with water, or form solvated species whenconcentrated in a solution with an appropriate organic solvent.

This invention also relates to pharmaceutical compositions comprising atherapeutically (or prophylactically) effective amount of thetranscriptional modulator, and a pharmaceutically acceptable carrier orexcipient. Carriers include e.g. saline, buffered saline, dextrose,water, glycerol, ethanol, and combinations thereof, and are discussed ingreater detail below. The composition, if desired, can also containminor amounts of wetting or emulsifying agents, or pH buffering agents.The transcriptional modulator can be a liquid solution, suspension,emulsion, tablet, pill, capsule, sustained release formulation, orpowder. The transcriptional modulator can be formulated as asuppository, with traditional binders and carriers such astriglycerides. Oral formulation can include standard carriers such aspharmaceutical grades of mannitol, lactose, starch, magnesium stearate,sodium saccharine, cellulose, magnesium carbonate, etc. Formulation mayinvolve mixing, granulating and compressing or dissolving theingredients as appropriate to the desired preparation.

The pharmaceutical carrier employed may be, for example, either a solidor liquid.

Illustrative solid carrier include lactose, terra alba, sucrose, talc,gelatin, agar, pectin, acacia, magnesium stearate, stearic acid and thelike. A solid carrier can include one or more substances which may alsoact as flavoring agents, lubricants, solubilizers, suspending agents,fillers, glidants, compression aids, binders or tablet-disintegratingagents; it can also be an encapsulating material. In powders, thecarrier is a finely divided solid which is in admixture with the finelydivided active ingredient. In tablets, the active ingredient is mixedwith a carrier having the necessary compression properties in suitableproportions, and compacted in the shape and size desired. The powdersand tablets preferably contain up to 99% of the active ingredient.Suitable solid carriers include, for example, calcium phosphate,magnesium stearate, talc, sugars, lactose, dextrin, starch, gelatin,cellulose, methyl cellulose, sodium carboxymethyl cellulose,polyvinylpyrrolidine,. low melting waxes and ion exchange resins.

Illustrative liquid carriers include syrup, peanut oil, olive oil,water, etc. Liquid carriers are used in preparing solutions,suspensions, emulsions, syrups, elixirs and pressurized compositions.The active ingredient can be dissolved or suspended in apharmaceutically acceptable liquid carrier such as water, an organicsolvent, a mixture of both or pharmaceutically acceptable oils or fats.The liquid carrier can contain other suitable pharmaceutical additivessuch as solubilizers, emulsifiers, buffers, preservatives, sweeteners,flavoring agents, suspending agents, thickening agents, colors,viscosity regulators, stabilizers or osmo-regulators. Suitable examplesof liquid carriers for oral and parenteral administration include water(partially containing additives as above, e.g. cellulose derivatives,preferably sodium carboxymethyl cellulose solution), alcohols (includingmonohydric alcohols and polyhydric alcohols, e.g. glycols) and theirderivatives, and oils (e.g. fractionated coconut oil and arachis oil).For parenteral administration, the carrier can also be an oily estersuch as ethyl oleate and isopropyl myristate. Sterile liquid carders areuseful in sterile liquid form compositions for parenteraladministration. The liquid carrier for pressurized compositions can behalogenated hydrocarbon or other pharmaceutically acceptable propellant.Liquid pharmaceutical compositions which are sterile solutions orsuspensions can be utilized by, for example, intramuscular,intraperitoneal or subcutaneous injection. Sterile solutions can also beadministered intravenously. The transcriptional modulator can also beadministered orally either in liquid or solid composition form.

The carrier or excipient may include time delay material well known tothe art, such as glyceryl monostearate or glyceryl distearate along orwith a wax, ethylcellulose, hydroxypropylmethylcellulose,methylmethacrylate and the like.

A wide variety of pharmaceutical forms can be employed. If a solidcarrier is used, the preparation can be tableted, placed in a hardgelatin capsule in powder or pellet form or in the form of a troche orlozenge. The amount of solid carrier will vary widely but preferablywill be from about 25 mg to about 1 g. If a liquid carrier is used, thepreparation will be in the form of a syrup, emulsion, soft gelatincapsule, sterile injectable solution or suspension in an ampule or vialor nonaqueous liquid suspension.

To obtain a stable water soluble dosage form, a pharmaceuticallyacceptable salt of a transcriptional modulator may be dissolved in anaqueous solution of an organic or inorganic acid, such as a 0.3 Msolution of succinic acid or citric acid. Alternatively, acidicderivatives can be dissolved in suitable basic solutions. If a solublesalt form is not available, the compound is dissolved in a suitablecosolvent or combinations thereof. Examples of such suitable cosolventsinclude, but are not limited to, alcohol, propylene glycol, polyethyleneglycol 300, polysorbate 80, glycerin, polyoxyethylated fatty acids,fatty alcohols or glycerin hydroxy fatty acids esters and the like inconcentrations ranging from 0-60% of the total volume.

Various delivery systems are known and can be used to administer thetranscriptional modulator, or the various formulations thereof,including tablets, capsules, injectable solutions, encapsulation inliposomes, microparticles, microcapsules, etc. Methods of introductioninclude but are not limited to dermal, intradermal, intramuscular,intraperitoneal, intravenous, subcutaneous, intranasal, pulmonary,epidural, ocular and (as is usually preferred) oral routes. The compoundmay be administered by any convenient or otherwise appropriate route,for example by infusion or bolus injection, by absorption throughepithelial or mucocutaneous linings (e.g., oral mucosa, rectal andintestinal mucosa, etc.) and may be administered together with otherbiologically active agents. Administration can be systemic or local. Fortreatment or prophylaxis of nasal, bronchial or pulmonary conditions,preferred routes of administration are oral, nasal or via a bronchialaerosol or nebulizer.

In certain embodiments, it may be desirable to administer thetranscriptional modulator locally to an area in need of treatment; thismay be achieved by, for example, and not by way of limitation, localinfusion during surgery, topical application, by injection, by means ofa catheter, by means of a suppository, or by means of a skin patch orimplant, said implant being of a porous, non-porous, or gelatinousmaterial, including membranes, such as sialastic membranes, or fibers.

In a specific embodiment, the transcriptional modulator is formulated inaccordance with routine procedures as a pharmaceutical compositionadapted for intravenous administration to human beings. Typically,compositions for intravenous administration are solutions in sterileisotonic aqueous buffer. Where necessary, the composition may alsoinclude a solubilizing agent and a local anesthetic to ease pain at theside of the injection. Generally, the ingredients are supplied eitherseparately or mixed together in unit dosage form, for example, as alyophilized powder or water free concentrate in a hermetically sealedcontainer such as an ampoule or sachette indicating the quantity ofactive agent. Where the composition is to be administered by infusion,it can be dispensed with an infusion bottle containing sterilepharmaceutical grade water or saline. Where the composition isadministered by injection, an ampoule of sterile water for injection orsaline can be provided so that the ingredients may be mixed prior toadministration.

Administration to an individual of an effective amount of the compoundcan also be accomplished topically by administering the compound(s)directly to the affected area of the skin of the individual. For thispurpose, the compound is administered or applied in a compositionincluding a pharmacologically acceptable topical carrier, such as a gel,an ointment, a lotion, or a cream, which includes, without limitation,such carriers as water, glycerol, alcohol, propylene glycol, fattyalcohols, triglycerides, fatty acid esters, or mineral oils.

Other topical carriers include liquid petroleum, isopropyl palmitate,polyethylene glycol, ethanol (95%), polyoxyethylene monolaurate (5%) inwater, or sodium lauryl sulfate (5%) in water. Other materials such asanti-oxidants, humectants, viscosity stabilizers, and similar agents maybe added as necessary. Percutaneous penetration enhancers such as Azonemay also be included.

In addition, in certain instances, it is expected that the compound maybe disposed within devices placed upon, in, or under the skin. Suchdevices include patches, implants, and injections which release thecompound into the skin, by either passive or active release mechanisms.

Materials and methods for producing the various formulations are knownin the art and may be adapted for practicing the subject invention. Seee.g. U.S. Pat. Nos. 5,182,293 and 4,837,311 (tablets, capsules and otheroral formulations as well as intravenous formulations) and EuropeanPatent Application Publication Nos. 0 649 659 (published Apr. 26, 1995;rapamycin formulation for IV administration) and 0 648 494 (publishedApr. 19, 1995; rapamycin formulation for oral administration).

The effective dose of the compound will typically be in the range ofabout 0.01 to about 50 mg/kgs, preferably about 0.1 to about 10 mg/kg ofmammalian body weight, administered in single or multiple doses.Generally, the compound may be administered to patients in need of suchtreatment in a daily dose range of about 1 to about 2000 mg per patient.In embodiments in which the transcriptional modulator includes a ligand.e.g., rapamycin or a derivative thereof, with some residualimmunosuppressive effects, it is preferred that the dose administered bebelow that associated with undue immunosuppressive effects.

The amount of compound which will be effective in the treatment orprevention of a particular disorder or condition will depend in part onthe nature of the disorder or condition, and can be determined bystandard clinical techniques. In addition, in vitro or in vivo assaysmay optionally be employed to help identify optimal dosage ranges.Effective doses may be extrapolated from dose-response curves derivedfrom in vitro or animal model test systems. The precise dosage levelshould be determined by the attending physician or other health careprovider and will depend upon well known factors, including route ofadministration, and the age, body weight, sex and general health of theindividual; the nature, severity and clinical stage of the disease; theuse (or not) of concomitant therapies; and the nature and extent ofgenetic engineering of cells in the patient.

The phrases "parenteral administration" and "administered parenterally"as used herein means modes of administration other than enteral andtopical administration, usually by injection, and includes, withoutlimitation, intravenous, intramuscular, intraarterial, intrathecal,intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal,transtracheal, subcutaneous, subcuticular, intraarticulare, subcapsular,subarachnoid, intraspinal and intrasternal injection and infusion.

The phrases "systemic administration," "administeredsystemically","peripheral administration" and "administeredperipherally" as used herein mean the administration of atranscriptional modulator, drug or other material, such that it entersthe subject's system and, thus, is subject to metabolism and other likeprocesses, for example, subcutaneous administration.

The invention also provides a pharmaceutical package or kit comprisingone or more containers filled with one or more of the ingredients of thepharmaceutical compositions of the invention. Optionally associated withsuch container(s) can be a notice in the form prescribed by agovernmental agency regulating the manufacture, use or sale ofpharmaceutical or biological products, which notice reflects approval bythe agency of manufacture, use or sale for human administration. Thepackage also can include instructions for using the transcriptionalmodulator within the methods of the invention.

Administration of Cells and Transcriptional Modulators

The genetically modified cells can be grown in culture under selectiveconditions and cells which are selected as having the construct may thenbe expanded and further analyzed, using, for example, the polymerasechain reaction for determining the presence of the construct in the hostcells. Once the modified host cells have been identified, they may thenbe used as planned, e.g. grown in culture or introduced into a hostorganism.

Depending upon the nature of the cells, the cells may be introduced intoa host organism, e.g. a mammal, in a wide variety of ways. Hematopoieticcells may be administered by injection into the vascular system, therebeing usually at least about 10⁴ cells and generally not more than about10¹⁰, more usually not more than about 10⁸ cells. The number of cellswhich are employed will depend upon a number of circumstances, thepurpose for the introduction, the lifetime of the cells, the protocol tobe used, for example, the number of administrations, the ability of thecells to multiply, the stability of the therapeutic agent, thephysiologic need for the therapeutic agent, and the like. Alternatively,with skin cells which may be used as a graft, the number of cells woulddepend upon the size of the layer to be applied to the burn or otherlesion. Generally, for myoblasts or fibroblasts, the number of cellswill at least about 10⁴ and not more than about 10⁸ and may be appliedas a dispersion, generally being injected at or near the site ofinterest. The cells will usually be in a physiologically-acceptablemedium.

Cells engineered in accordance with this invention may also beencapsulated, e.g. using conventional biocompatible materials andmethods, prior to implantation into the host organism or patient for theproduction of a therapeutic protein. See e.g. Hguyen et al, TissueImplant Systems and Methods for Sustaining viable High Cell Densitieswithin a Host, U.S. Pat. No. 5,314,471 (Baxter International, Inc.);Uludag and Sefton, 1993, J Biomed. Mater. Res. 27(10):1213-24 (HepG2cells/hydroxyethyl methacrylate-methyl methacrylate membranes); Chang etal, 1993, Hum Gene Ther 4(4):433-40 (mouse Ltk-cells expressinghGH/immunoprotective perm-selective alginate microcapsules; Reddy et al,1993, J Infect Dis 168(4):1082-3 (alginate); Tai and Sun, 1993, FASEB J7(11):1061-9 (mouse fibroblasts expressinghGH/alginate-poly-L-lysine-alginate membrane); Ao et al, 1995,Transplantaion Proc. 27(6):3349, 3350 (alginate); Rajotte et al, 1995,Transplantation Proc. 27(6):3389 (alginate); Lakey et al, 1995,Transplantation Proc. 27(6):3266 (alginate); Korbutt et al, 1995,Transplantation Proc. 27(6):3212 (alginate); Dorian et al, U.S. Pat. No.5,429,821 (alginate); Emerich et al, 1993, Exp Neurol 122(1):37-47(polymer-encapsulated PC12 cells); Sagen et al, 1993, J Neurosci13(6):2415-23 (bovine chromaffin cells encapsulated in semipermeablepolymer membrane and implanted into rat spinal subarachnoid space);Aebischer et al, 1994, Exp Neurol 126(2):151-8 (polymer-encapsulated ratPC12 cells implanted into monkeys; see also Aebischer, WO 92/19595);Savelkoul et al, 1994, J Immunol Methods 170(2):185-96 (encapsulatedhybridomas producing antibodies; encapsulated transfected cell linesexpressing various cytokines); Winn et al, 1994, PNAS USA 91(6):2324-8(engineered BHK cells expressing human nerve growth factor encapsulatedin an immunoisolation polymeric device and transplanted into rats);Emerich et al, 1994, Prog Neuropsychopharmacol Biol Psychiatry18(5):935-46 (polymer-encapsulated PC12 cells implanted into rats);Kordower et al, 1994, PNAS USA 91(23):10898-902 (polymer-encapsulatedengineered BHK cells expressing hNGF implanted into monkeys) and Butleret al WO 95/04521 (encapsulated device). The cells may then beintroduced in encapsulated form into an animal host, preferably a mammaland more preferably a human subject in need thereof. Preferably theencapsulating material is semipermeable, permitting release into thehost of secreted proteins produced by the encapsulated cells. In manyembodiments the semipermeable encapsulation renders the encapsulatedcells immunologically isolated from the host organism in which theencapsulated cells are introduced. In those embodiments the cells to beencapsulated may express one or more chimeric proteins containingcomponent domains derived from proteins of the host species and/or fromviral proteins or proteins from species other than the host species. Forexample in such cases the chimeras may contain elements derived fromGAL4 and VP16. The cells may be derived from one or more individualsother than the recipient and may be derived from a species other thanthat of the recipient organism or patient.

Instead of ex vivo modification of the cells, in many situations one maywish to modify cells in vivo. For this purpose, various techniques havebeen developed for modification of target tissue and cells in vivo. Anumber of virus vectors have been developed, such as adenovirus andretroviruses, which allow for transfection and random integration of thevirus into the host. See, for example, Dubensky et al. (1984) Proc.Natl. Acad. Sci. USA 81, 7529-7533; Kaneda et al., (1989) Science243,375-378; Hiebert et al. (1989) Proc. Natl. Acad. Sci. USA 86,3594-3598; Hatzoglu et al. (1990) J. Biol. Chem. 265, 17285-17293 andFerry, et al. (1991) Proc. Natl. Acad. Sci. USA 88, 8377-8381. Thevector may be administered by injection, e.g. intravascularly orintramuscularly, inhalation, or other parenteral mode.

In accordance with in vivo genetic modification, the manner of themodification will depend on the nature of the tissue, the efficiency ofcellular modification required, the number of opportunities to modifythe particular cells, the accessibility of the tissue to the DNAcomposition to be introduced, and the like. By employing an attenuatedor modified retrovirus carrying a target transcriptional initiationregion, if desired, one can activate the virus using one of the subjecttranscription factor constructs, so that the virus may be produced andtransfect adjacent cells.

The DNA introduction need not result in integration in every case. Insome situations, transient maintenance of the DNA introduced may besufficient. In this way, one could have a short term effect, where cellscould be introduced into the host and then turned on after apredetermined time, for example, after the cells have been able to hometo a particular site.

Depending upon the binding affinity of the transcriptional modulator,the response desired, the manner of administration, the half-life, thenumber of cells present, various protocols may be employed. Thetranscriptional modulator may be administered parenterally or orally.The number of administrations will depend upon the factors describedabove. The transcriptional modulator may be taken orally as a pill,powder, or dispersion; bucally; sublingually; injected intravascularly,intraperitoneally, subcutaneously; by inhalation, or the like. Thetranscriptional modulator (and free monomeric antagonist) may beformulated using convenitonal methods and materials well known in theart for the various routes of administration. The precise dose andparticular method of administration will depend upon the above factorsand be determined by the attending physician or human or animalhealthcare provider. For the most part, the manner of administrationwill be determined empirically.

In the event that the activation by the transcriptional modulator is tobe reversed, the free monomeric compound may be administered or othersingle binding site compound which can compete with the transcriptionalmodulator. Thus, in the case of an adverse reaction or the desire toterminate the therapeutic effect, the monomeric binding compound can beadministered in any convenient way, particularly intravascularly, if arapid reversal is desired. Alternatively, one may provide for thepresence of an inactivation domain with a DNA binding domain, orapoptosis by having Fas or TNF receptor present as constitutivelyexpressed constructs.

The particular dosage of the transcriptional modulator for anyapplication may be determined in accordance with the procedures used fortherapeutic dosage monitoring, where maintenance of a particular levelof expression is desired over an extended period of times, for example,greater than about two weeks, or where there is repetitive therapy, withindividual or repeated doses of transcriptional modulator over shortperiods of time, with extended intervals, for example, two weeks ormore. A dose of the transcriptional modulator within a predeterminedrange would be given and monitored for response, so as to obtain atime-expression level relationship, as well as observing therapeuticresponse. Depending on the levels observed during the time period andthe therapeutic response, one could provide a larger or smaller dose thenext time, following the response. This process would be iterativelyrepeated until one obtained a dosage within the therapeutic range. Wherethe transcriptional modulator is chronically administered, once themaintenance dosage of the transcriptional modulator is determined, onecould then do assays at extended intervals to be assured that thecellular system is providing the appropriate response and level of theexpression product.

The subject methodology and compositions may be used for the treatmentof a wide variety of conditions and indications. For example, B- andT-cells may be used in the treatment of cancer, infectious diseases,metabolic deficiencies, cardiovascular disease, hereditary coagulationdeficiencies, autoimmune diseases, joint degenerative diseases, e.g.arthritis, pulmonary disease, kidney disease, endocrine abnormalities,etc. Various cells involved with structure, such as fibroblasts andmyoblasts, may be used in the treatment of genetic deficiencies, such asconnective tissue deficiencies, arthritis, hepatic disease, etc.Hepatocytes could be used in cases where large amounts of a protein mustbe made to complement a deficiency or to deliver a therapeutic productto the liver or portal circulation.

Applications

1. Regulated gene therapy. In many instances, the ability to switch atherapeutic gene on and off at will or the ability to titrate expressionwith precision are important for therapeutic efficacy. This invention isparticularly well suited for achieving regulated expression of atherapeutic target gene in the context of human gene therapy. Asdescribed in detail in the section entitled "Transcriptional ModulatingPortion",the transcriptional modulators of the present invention can actin a genetically engineered cell (e.g., a cell genetically modified tocontain at least one chimeric protein and/or at least one target gene)by one or more of (i) a direct or indirect interaction(s) of thetranscriptional modulator with a component of the transcriptionalmachinery; (ii) an alteration(s) of chromatin structure; and (iii) analteration(s) in the effective concentration of the transcriptionalmodulator in the nucleus of a cell. Contacting the engineered cells orthe progeny thereof with the transcriptional modulators (byadministering the agent to the subject) leads to expression of thetarget gene. In practice, the level of target gene expression should bea function of the number or concentration of chimericprotein-transcriptional modulator complexes, which should in turn be afunction of the concentration of the transcriptional modulator.

The transcriptional modulator may be administered to the patient asdesired to activate transcription of the target gene. Depending upon thebinding affinity and activity of the transcriptional modulator, theresponse desired, the manner of administration, the half-life, thenumber of cells present, various protocols may be employed. Thetranscriptional modulator may be administered parenterally or orally.The number of administrations will depend upon the factors describedabove. The ligand may be taken orally as a pill, powder, or dispersion;bucally; sublingually; injected intravascularly, intraperitoneally,intramuscularly, subcutaneously; by inhalation, or the like. The ligand(or antagonist) may be formulated using conventional methods andmaterials known in the art for the various routes of administration. Theprecise dose and particular method of administration will depend uponthe above factors and be determined by the attending physician. For themost part, the manner of administration will be determined empirically.

In the event that transcriptional activation by the transcriptionalmodulator is to be reversed or terminated, compound which can competewith the transcriptional modulator may be administered. Alternatively,combinations of the transcriptional modulators of the present inventioncan be used to control gene expression. For example, combinations oftranscriptional activators containing a transcriptional activatorportion or a transcriptional repressor portion can be used. Thus, in thecase of an adverse reaction or the desire to terminate the therapeuticeffect, an antagonist to the transcriptional modulator can beadministered in any convenient way, particularly intravascularly, if arapid reversal is desired. In another approach, cells may be eliminatedthrough apoptosis via signalling through Fas or TNF receptor asdescribed elsewhere. See International Patent ApplicationsPCT/US94/01617 and PCT/US94/08008.

The particular dosage of the transcriptional modulator for anyapplication may be determined in accordance with the procedures used fortherapeutic dosage monitoring, where maintenance of a particular levelof expression is desired over an extended period of times, for example,greater than about two weeks, or where there is repetitive therapy, withindividual or repeated doses of transcriptional modulator over shortperiods of time, with extended intervals, for example, two weeks ormore. A dose of the transcriptional modulator within a predeterminedrange would be given and monitored for response, so as to obtain atime-expression level relationship, as well as observing therapeuticresponse. Depending on the levels observed during the time period andthe therapeutic response, one could provide a larger or smaller dose thenext time, following the response. This process would be iterativelyrepeated until one obtained a dosage within the therapeutic range. Wherethe ligand is chronically administered, once the maintenance dosage ofthe ligand is determined, one could then do assays at extended intervalsto be assured that the cellular system is providing the appropriateresponse and level of the expression product.

2. Production of recombinant proteins and viruses. Production ofrecombinant therapeutic proteins for commercial and investigationalpurposes is often achieved through the use of mammalian cell linesengineered to express the protein at high level. The use of mammaliancells, rather than bacteria or yeast, is indicated where the properfunction of the protein requires post-translational modifications notgenerally performed by heterologous cells. Examples of proteins producedcommercially this way include erythropoietin, tissue plasminogenactivator, clotting factors such as Factor VIII:c, antibodies, etc. Thecost of producing proteins in this fashion is directly related to thelevel of expression achieved in the engineered cells. A secondlimitation on the production of such proteins is toxicity to the hostcell: Protein expression may prevent cells from growing to high density,sharply reducing production levels. Therefore, the ability to tightlycontrol protein expression, as described for regulated gene therapy,permits cells to be grown to high density in the absence of proteinproduction. Only after an optimum cell density is reached, is expressionof the gene activated and the protein product subsequently harvested.

A similar problem is encountered in the construction and use of"packaging lines" for the production of recombinant viruses forcommercial (e.g., gene therapy) and experimental use. These cell linesare engineered to produce viral proteins required for the assembly ofinfectious viral particles harboring defective recombinant genomes.Viral vectors that are dependent on such packaging lines includeretrovirus, adenovirus, and adeno-associated virus. In the latter case,the titer of the virus stock obtained from a packaging line is directlyrelated to the level of production of the viral rep and core proteins.But these proteins are highly toxic to the host cells. Therefore, it hasproven difficult to generate high-titer recombinant AAV viruses. Thisinvention provides a solution to this problem, by allowing theconstruction of packaging lines in which the rep and core genes areplaced under the control of regulatable transcription factors of thedesign described here. The packaging cell line can be grown to highdensity, infected with helper virus, and transfected with therecombinant viral genome. Then, expression of the viral proteins encodedby the packaging cells is induced by the addition of dimerizing agent toallow the production of virus at high titer.

3. Biological research. This invention is applicable to a wide range ofbiological experiments in which precise control over a target gene isdesired. These include: (1) expression of a protein or RNA of interestfor biochemical purification; (2) regulated expression of a protein orRNA of interest in tissue culture cells (or in vivo, via engineeredcells) for the purposes of evaluating its biological function; (3)regulated expression of a protein or RNA of interest in transgenicanimals for the purposes of evaluating its biological function; (4)regulating the expression of a gene encoding another regulatory protein,ribozyme or antisense molecule that acts on an endogenous gene for thepurposes of evaluating the biological function of that gene. Transgenicanimal models and other applications in which the components of thisinvention may be adapted include those disclosed in PCT/US95/10591.

This invention further provides kits useful for the foregoingapplications. Such kits contain DNA constructs encoding and capable ofdirecting the expression of chimeric proteins of this invention and, inembodiments involving regulated gene transcription, a target geneconstruct containing a target gene linked to one or more transcriptioalcontrol elements which are activated by the transcriptionalmodulator-chimeric protein complex. Alternatively, the target geneconstruct may contain a cloning site for insertion of a desired targetgene by the practitioner. Such kits may also contain a sample of a smallmolecule of transcriptional modulator, or any combination oftranscriptional modulators.

The practice of the present invention employs, unless otherwiseindicated, conventional techniques of cell biology, cell culture,molecular biology, transgenic biology, microbiology, recombinant DNA,and immunology, which are within the skill of the art. Such techniquesare explained fully in the literature. See, for example, MolecularCloning A Laboratory Manual, 2nd Ed., ed. by Sambrook, Fritsch andManiatis (Cold Spring Harbor Laboratory Press: 1989); DNA Cloning,Volumes I and II (D. N. Glover ed., 1985); Oligonucleotide Synthesis (M.J. Gait ed., 1984); Mullis et al. U.S. Pat. No: 4,683,195; Nucleic AcidHybridization (B. D. Hames & S. J. Higgins eds. 1984); Transcription AndTranslation (B. D. Hames & S. J. Higgins eds. 1984); Culture Of AnimalCells (R. I. Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells AndEnzymes (IRL Press, 1986); B. Perbal, A Practical Guide To MolecularCloning (1984); the treatise, Methods In Enzymology (Academic Press,Inc., N.Y.); Gene Transfer Vectors For Mammalian Cells (J. H. Miller andM. P. Calos eds., 1987, Cold Spring Harbor Laboratory); Methods InEnzymology, Vols. 154 and 155 (Wu et al. eds.), Immunochemical MethodsIn Cell And Molecular Biology (Mayer and Walker, eds., Academic Press,London, 1987); Handbook Of Experimental Immunology, Volumes I-IV (D. M.Weir and C. C. Blackwell, eds., 1986); Manipulating the Mouse Embryo,(Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986).

EXAMPLES

The invention now being generally described, it will be more readilyunderstood by reference to the following examples which are includedmerely for purposes of illustration of certain aspects and embodimentsof the present invention, and are not intended to limit the invention.

Synthesis of Transcriptional Modulators (Non-natural Coactivator L-1 andD-1)

The synthesized transcriptional modulators and techniques describedbelow were used in the Examples.

The Boc-protected hexamethylenediamine carbamate derivative of FK506 wasdeprotected and N-bromoacetylated in situ by treatment with bromoaceticanhydride as described in Robey, F. A. and Fields, R. L., Anal.Biochem., 177:373-377 (1989). The product was purified by flashchromatography and its structure was confirmed by fast atombombardment-HRMS. The L and D activator peptidesCGSDALDDFDLDMLGSDALD-DFDLDMLGS-NH₂ (SEQ ID NO:1) were synthesized bystandard solid phase peptide synthesis (rink resin)/deprotectionmethods, purified by reversed-phase HPLC, and characterized by aminoacid analysis and HRMS. The bromoacetylated FK506 derivative andactivator peptide were coupled using a procedure reported for proteinligation [Muir, T. W., et al., Biochemistry, 33:7702-7708 (1994)].Briefly, the activator peptide (650 μg, 0.21 μmol) was combined withbromoacetylated-FK506 (250 μg 0.23 μmol) in 300 μl of 95%dimethyl-formamide/5% 0.1M sodium phosphate buffer (pH 7) and thereaction was allowed to proceed overnight at room temperature. Theproduct was purified by anion-exchange chromatography on a WatersGen-Pack FAX HPLC column (4.6×100 mm) using a gradient of 5-45% B over40 min (eluent A: 25 mM Tris-HCl, pH 7.5/10% Ch₃ CN; eluent B: eluentA+1.0 M NaCl). After desalting on a C₁₈ Waters Sep-Pack Cartridge, theproduct was eluted with 9:1 acetonitrile/water and lyophilized. L-1 andD-1 conjugates were quantified by amino acid analysis and characterizedby electrospray-ionization mass spectroscopy (negative ion absorptionmode).

In Vitro Transcription. HeLa nuclear extracts were prepared as describedin Dignam, J. D., et al., Nucleic Acids Res., 11: 1475-1489 (1983).GAL4(1-147)-VP16(413-490) was overexpressed and purified as described[Chasman, D. J., et al., Mol. Cell. Biol., 9:4746-4749 (1989)]. Theexpression vectors coding for GAL4(1-94), and GAL4(1-94). FKBP12 weresub-cloned into p1.M1 [Sodcoka, M., et al., Bioorganic. Med. Chem.Lett., 3:1089-1094 (1993)] and the resulting fusion proteins wereoverexpressed and purified to homogeneity essentially as described forGAL4(1-147)-VP16(413-490). In vitro transcription assays were performedas described in Carey, M., et al., Science, 247:710-712 (1990). Themixtures contained 25 μl of HeLa nuclear extract (3.15 mg/ml) in DignamD buffer (20 mM Hepes, pH 7.9/100 mM KCl/20% glycerol/0.2 mM EDTA/0.5 mMDTT/0.5 mM phenylmethylsufonyl fluoride), 8 mM MgCl₂, 10 mM ammoniumsulfate, 1% PEG 8000, 0.1 mg/ml BSA, 8 units RNA guard, 200 ng of 1% PEG8000, 0.1 mg/ml BSA, 8 units RNA guard, 200 ng of pGEM3 as carrier, 30ng of template pG₅ E4T and either no GAL4 protein or an amountsufficient to give >90% protein-DNA complex, as determined byindependent gel-shift assays. The optimal amount of compounds L-1 andD-1 was titrated by transcription in vitro. GAL4-FKBP was preincubated10 min at room temperature with 10 molar equivalents of coactivator L-1or D-1 followed by a 10 min incubation time with the reporter template.After addition of the nuclear extract and further preincubation for 15min at room temperature, the reaction was initiated by addition of 2 μlof rNTP mix 10 mM. After 1 h at 30° C., the reaction was terminated andthe reaction products were purified and analyzed by primer extension asdescribed [Carey, M., et al., Science, 247:710-712 (1990)]. Eachexperiment was repeated a minimum of three times.

In Vitro Transcription. Jurkat cells were maintained in RPMI 1640 mediacontaining 10% (vol/vol) calf serum, L-glutamine and 1% (vol/vol)penicillin/streptomycin. Cells plated in a six-well tissue culture plate(2×10⁶ cells per well) were transfected (6 μl DMRIE-C; GIBCO/BRL) with 2μg each of G5IL2SX and GF₃. After 24 h incubation, the medium wasremoved and the cells were resuspended in fresh Opti-Mem I reduced serummedium and atiquoted into a 96-well microtiter plate (2×10⁶ cells perwell). Various concentrations of L-1 and D-1 in DMRIE-C were added tothe cells. After 24 h, aliquots were removed and assayed for secretedalkaline phosphatase (SEAP) activity as described in Belahaw, P. J., etal., Proc. Natl. Acad. Sci. USA, 93:4604-4607 (1996). In competitionexperiments, 1 μM rapamycin was added at the same time as D-1.

A molecule designed to serve as an intermediary between a DNA-bindingprotein and the transcriptional apparatus should incorporate bindingelements for each of these two macromolecular targets. In order to meetthis criterion, the immunosuppresive drug FK506, which binds with highaffinity (K_(d) =0.4 nM) to the immunophilin FK binding protein 12(FKBP12) was chosen [Standaert, R. F., et al., Nature (London),346:671-674 (1990). FK506 and certain derivatives thereof can betargeted to the DNA binding domain of GAL4 by fusing the GAL4 domain toFKBP (GAL4-FKBP) [Belahaw, P. J., et al., Proc. Natl. Acad. Sci. USA,93:4604-4607 (1996)]. Modification of the calcineurin-binding surface ofFK506 yields derivatives that lack immunosuppressive properties butretain the ability to bind FKBP with high affinity [Belahaw, P. J., etal., Proc. Natl. Acad. Sci. USA, 93:4604-4607 (1996); Bierer, B. E., etal., Science, 250:556-559 (1990)]. The nonimmunosuppressive FK506derivative, bearing a hydroxyethyl group at C-21, was further equippedwith an activator element through the addition of a linker, to which atranscriptional activation domain was attached. In particular, a29-amino acid L peptide containing a tandemly repeated undecamersequence derived from the N-terminal portion of the VP16 activationdomain was attached (FIGS. 1A and B). This L peptide, when directlyfused to the GAL4 DNA-binding domain, is a potent activator oftranscription in vivo [Seipel, K., et al., EMBO J., 11:4961-4968(1992)], most likely through binding directly to component(s) of thebasal transcriptional apparatus [Lin, Y.-S, et al., Nature (London),353:569-571 (1991)]. Thus, the FK506-peptide conjugate L-1 could inprinciple be capable of bridging GAL4-FKBP and the basal transcriptionalapparatus (FIG. 1C).

EXAMPLE I In Vitro Modulation of Transcription Using TranscriptionalModulators

To assess the ability of L-1 to function as a transcriptionalcoactivator, in vitro transcription assays using HeLa nuclear extractsand a reporter gene pG₅ E4T containing 5 GAL4 sites upstream of anadenovirus E4 promoter were carried out (FIGS. 2-3B). As shown in FIG.2B (lane 5), L-1 stimulated transcription in the presence of GAL4-FKBP,but was unable to stimulate in the absence of GAL4-FKBP (lane 6). Theactivation potential of L-1 was significantly reduced in the presence ofadded rapamycin or GST-FKBP. These molecules are known to compete withthe L-1/GAL4-FKBP interaction [Bierer, R. F., et al., Proc. Natl. Acad.Sci. USA, 87:9231-9235 (1990)]. The competition assays were carried outas described (Carey, M. et al. (1990) Science 247, 710-712), exceptGAL4-FKBP was preincubated 10 min at room temperature with 10equivalents of compound L-1 or D-1 plus 100 equivalents of competitor(rapamycin), prior to the addition of the reporter template and the HeLanuclear extract. FKBP12 was expressed in Escherichia coli as aglutathione 5-transferase fusion protein and purified on glutathionagarose beads. These experiments demonstrate that L-1 functions as acoactivator with GAL4-FKBP in vitro.

Acyclic peptides having the natural L stereochemical configuration arehighly susceptible to proteolysis, in vivo, especially when they possessunmodified amide bonds [Saffran, M., et al., Science, 233:1081-1084(1986)]. For this reason, it seemed unlikely that L-1 would functioneffectively to activate transcription in cells. On the other hand,peptides bearing the nonnatural D stereochemistry are often resistant toproteolysis, even in linear form [Wermuth, J., et al., J. Am. Chem.Soc., 119:1328-1335 (1997)]. However, it is unknown whether a Dconfigured peptide, or for that matter any nonnatural ligand, canfunction as a transcriptional activator. To test this whether a Dconfigured peptide can function as a transcriptional activator, an FK506conjugate bearing the enantiomeric, D configured version of the VP16activation peptide was used. This conjugate is referred to herein as D-1(FIGS. 1B and C). The results of in vitro transcription assaysdemonstrate that D-1 reproducibly stimulated transcription to asignificant extent, though to a slightly lesser extent than L-1 (FIG. 2,lane 7). D-1 exhibited no activation in the absence of GAL4-FKBP (lane8), and its activation potential was significantly reduced in thepresence of added rapamycin or gluthione S-transferase-FKBP. Theseresults establish that the nonnatural molecule D-1 functions effectivelyas a transcriptional coactivator in vitro.

EXAMPLE II In Vivo Modulation of Transcription Using a SyntheticTranscriptional Modulators

To determine whether D-1 can function as a coactivator of transcriptionin living cells, Jurkat cells were transiently cotransfected with (i) areporter plasmid containing the SEAP cDNA and an interleukin 2 promoterwith five upstream GAL4 DNA binding sites, and (ii) a constitutiveexpression plasmid encoding the GAL4 DNA-binding domain fused to threetandemly repeated FKBP12s modules (GAL4-FKBP3, FIG. 3). The transfectedcells were subsequently treated with various concentrations of L-1 andD-1 incorporated into liposomes to enhance cell-permeability. When thecells were treated with D-1, expression of the SEAP reporter gene wasstimulated in a dose-dependent manner (FIG. 3B). When the GAL4-FKBP3expression plasmid was omitted during the transfection step, the cellswere unresponsive to D-1, indicating that the activation stimulus wasdependent upon a interaction between GAL4-FKBP3 and D-1. Consistent withthis, rapamycin abolished the activation signal, presumably by competingwith the FK506 portion of D-1 for the FKBP domain in GAL4-FKBP3. Bycontrast, L-1 showed no detectable ability to activate SEAP expressionin transfected cells (FIG. 3B). As the L activation peptide is known tostimulate transcription in HeLa [Seipel, K., et al., EMBO J.,11:4961-4968 (1992)] and Jurkat cells when fused to the GAL4DNA bindingdomain, and as FK506 derivatives are capable of saturating FKBP bindingsites under the conditions of these experiments [Belshaw, P. J., et al.,Proc. Natl. Acad Sci. USA, 93:4604-4607 (1996); Ho, S. H., et al.,Nature (London), 382:822-826 (1996)]. The failure of L-1 to activatetranscription seems most likely to have resulted from intracellularproteolysis. The nuclear extracts used in vitro transcription assaysalmost certainly contain proteases as well; however, these arepresumably rendered inactive by the proteases inhibitors included in theassays.

The results described herein show that an approximately 4 kDa syntheticmolecule containing two linked binding elements, one that targets aDNA-binding protein and another that targets the transcriptionalmachinery, can coactivate transcription of a mammalian promoter.Specifically, it has been shown that a designed coactivator containing anonnatural completely D configured peptide stimulates transcription invitro with only slightly less potency than the corresponding coactivatorbearing the natural L configuration. Strikingly, the nonnatural moleculeD-1 also stimulates transcription of a GAL4-driven promoter in vivo,when present in conjunction with GAL4-FKBP. The examples describedherein thus demonstrate both the feasibility of using small molecules tocoactivate gene expression in vitro and in vivo, and the ability ofcompletely nonnatural small molecules to serve this function.

The description of possible mechanisms set forth below is not intendedto be limiting of the invention. The ability of a D configured peptideto serve as an activation domain raises a number of interestingmechanistic issues, such as whether the L and D peptides contact thesame target. As the FK506 portion of either L-l or D-1 almost certainlyinteracts much more strongly with its target than does the activatorpeptide portion, it is conceivable that the synthetic coactivators firstform a stable complex with the GAL4-FKBP fusion protein, and theresulting DNA-bound complex then recruits the transcriptional machineryto the promoter through direct peptide-protein contacts. The target ofthe L activator peptide is likely to be TFIIB, if indeed it contacts thesame protein as the N-terminal portion of the VP16 activation domain,from which the 29-mer is derived [Seipel, K., et al., EMBO J,11:4961-4968 (1992); Lin, Y.-S, et al., Nature (London), 353:569-571(1991)]. Regardless whether the peptide is fused directly to aDNA-binding domain or bound noncovalently through the aegis ofFK506-FKBP interactions, the target of the L peptide probably remainsthe same. No high-resolution structural information is available for anyactivation domain bound to its target. However, it has recently beendemonstrated that an activation peptide derived from the C-terminaldomain of VP16 folds into an a-helix upon interaction with its target.TAFπ31, with nonpolar contacts being made by hydrophobic amino acidside-chains that lie among one face of the helix, including one keycontact made by a phenylalanine residue that apparently represents acommon feature of several acidic activation domains [Uesugi, M., et al.,Science, in press (1997)]. It is noted that the repeated sequence in theactivation peptide not only contains a Phe residue that is known toserve as important functional role in the intact VP16 activation domain[Cress, W. D. and Triezenberg, S. J., Science, 251:87-90 (1991)], butalso contains additional hydrophobic residues at the i+3 and i+4 and i+5positions, which would all lie along one face of a putative α-helix. Ifindeed the L peptide contacts its transcriptional target using theseresidues, it is conceivable that the D peptide could make similarcontacts, (though in reversed orientation with respect to the target),because the indicated nonpolar residues would all lie along one face ofa D helix, and hydrophobic interactions can exhibit remarkable stericand geometric plasticity. Consistent with this notion, L and Dconfigured calmodulin-binding peptides having the same amino acidsequence bind with similar strength to calmodulin [Fisher, P. J., etal., Nature (London), 368:651-653 (1994). Of course, it remains a realpossibility that the L-1 and D-1 target different components of thetranscriptional apparatus.

The present demonstration that a nonnatural entity can activatetranscription, together with prior findings that activators arise at asmall but significant frequency in libraries of random fusion peptides[Ma, J. and Plashne, M., Cell, 51:113-119 (1980)], indicates that it ispossible to identify activation domains with low molecular weight fromcombinatorial libraries of organic molecules. A major limitation to anysuch screening effort has been the requirement that a prospectiveactivator be physically associated with a DNA-binding domain. The systemdescribed herein provides a means of overcoming this limitation bylinking the activator to a membrane-permeant organic ligand, e.g.,FK506.

EXAMPLE III Preparation Of A Combinatorial Test Compound Library

A library of test compounds is prepared as follows:

Resin beads (Merrifield resin) are divided into 5 aliquots, and eachaliquot is placed in a reaction vessel of an automatic peptidesynthesizer. To each reaction vessel is added one of 59-fluorenylmethoxycarbonyl-(Fmoc) protected amino acids (available from,e.g., Sigma Chemical, St. Louis, Mo.), and the protected amino acids areallowed to react with the resin to provide amino acid-derivatizedresins. The aliquots are then washed to remove excess reagents andimpurities. Each aliquot is treated with piperidine to remove the Fmocprotecting group and the beads are again washed to remove reagents andimpurities. Each aliquot of beads is further divided into five aliquots,and each aliquot is treated with an Fmoc-protected pentafluorophenylamino acid ester (Sigma). After reaction, the beads are washed to removeimpurities, and again deprotected and split into five further aliquots,each of which is treated with one of five activated amino acids. Afterreaction, washing, and deprotection, a library of 125 tripeptides isobtained. Each of the tripeptides is then coupled (at the N-terminus) toa tandemly repeated undecamer sequence derived from the N-terminal ofthe VP16 activation domain, as described herein. Each peptide is thentreated with an activated ester of cysteine to provide an N-terminalcysteine residue. The resulting resin-bound library includes 125peptides differing at the C-terminal tripeptide.

The resin-bound peptide library is then coupled to a bromoacetylatedderivative of FK506 (e.g., as described herein) to provide a library oftest compounds having an FK506 portion and a test transcriptionalmodulating portion. These test compounds are then screened to determinethe effect of C-terminal substitution on the transcriptional modulatingactivity of the peptide and transcriptional modulators are identified.

The contents of all cited references (including literature references,issued patents, published patent applications, and co-pending patentapplications) cited throughout this application (including theBackground Section) are hereby expressly incorporated by reference. Theentire contents of Appendix A entitled "A Nonnatural TranscriptionalCoactivator" also is incorporated by reference.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                   - -  - - <160> NUMBER OF SEQ ID NOS: 1                                        - - <210> SEQ ID NO 1                                                        <211> LENGTH: 29                                                              <212> TYPE: PRT                                                               <213> ORGANISM: synthetic construct                                            - - <400> SEQUENCE: 1                                                         - - Cys Gly Ser Asp Ala Leu Asp Asp Phe Asp Le - #u Asp Met Leu Gly        Ser                                                                               1               5 - #                 10 - #                 15             - - Asp Ala Leu Asp Asp Phe Asp Leu Asp Met Le - #u Gly Ser                               20     - #             25                                       __________________________________________________________________________

We claim:
 1. A method for identifying a transcriptional modulator,comprising:providing a cell comprising (i) a genetic construct encodinga chimeric protein which comprises at least one ligand-binding domainand a DNA-binding domain which is heterologous thereto, wherein theligand-binding domain binds to a selected ligand, and (ii) a target geneunder the control of at least one transcriptional regulatory elementwhich is recognized by the DNA-binding domain of the chimeric protein;contacting the cell with a test compound which comprises the selectedligand linked to a test transcriptional modulating portion underconditions which allow transcription to occur; and detecting any changesin transcriptional activity of the target gene in the presence of thetest compound relative to that detected in the absence of the testcompound, wherein a change in the level of transcriptional activity ofthe target gene detected in the presence of the test compound relativeto that detected in the absence of the test compound indicates that thetest compound is a transcriptional modulator.
 2. A method foridentifying a transcriptional modulator, comprising:providing a reactionmixture under conditions which allow transcription to occur, saidreaction mixture including,(i) a chimeric protein which comprises atleast one ligand-binding domain and a DNA-binding domain which isheterologous thereto, wherein the ligand-binding domain binds to aselected ligand; (ii) a target gene under the control of at least onetranscriptional regulatory element which is recognized by theDNA-binding domain of the chimeric protein; (iii) a cell-freetranscription system; (iv) a test compound which comprises the selectedligand linked to a test transcriptional modulating portion; anddetecting any changes in transcriptional activity of the target gene inthe presence of the test compound relative to that detected in theabsence of the test compound,wherein a change in the level oftranscriptional activity of the target gene detected in the presence ofthe test compound relative to that detected in the absence of the testcompound indicates that the test compound is a transcriptionalmodulator.
 3. The method of claim 1 or 2, wherein the transcriptionalmodulating portion is a chemical moiety and wherein the test compoundhas a molecular weight of less than about 3 kD.
 4. The method of claim 1or 2, wherein the transcriptional modulating portion is a chemicalmoiety and wherein the test compound has a molecular weight of less thanabout 1.5 kD.
 5. A method for identifying a transcriptional modulatorfrom a plurality of test compounds, comprising:providing cellscomprising,(i) a genetic construct encoding a chimeric protein whichcomprises at least one ligand-binding domain and a DNA-binding domainwhich is heterologous thereto, wherein the ligand-binding domain bindsto a selected ligand; (ii) a target gene under the control of at leastone transcriptional regulatory element which is recognized by theDNA-binding domain of the chimeric protein; contacting an aliquot of thecells with one or more test compounds, each of which comprises theselected ligand linked to at least one of a plurality of testtranscriptional modulating portions under conditions which allowtranscription to occur; and detecting any changes in transcriptionalactivity of the target gene in the presence of a given test compoundrelative to that detected in the absence of the test compound,wherein achange in the level of transcriptional activity of the target genedetected in the presence of the test compound relative to that detectedin the absence of the test compound indicates that the test compound isa transcriptional modulator.
 6. The method of any of claims 1, 2 or 5,wherein the transcriptional modulating portion is a chemical moiety. 7.The method of any of claims 1, 2 or 5, wherein the transcriptionalmodulating portion is a proteinaceous domain.
 8. The method of either ofclaims 1 or 5, wherein the chimeric protein is in the nucleus of thecell.
 9. The method of either of claims 1 or 5, wherein the chimericprotein is in the cytoplasm of the cell.
 10. The method of claim 5,wherein the transcriptional modulating portion is a chemical moiety, andwherein one or more of the test compounds has a molecular weight of lessthan about 3 kD.
 11. The method of claim 5, wherein the transcriptionalmodulating portion is a chemical moiety, and wherein one or more of thetest compounds has a molecular weight of less than about 1.5 kD.
 12. Themethod of claim 6, wherein the test compound is membrane-permeant. 13.The method of claim 6, wherein the transcriptional modulating portion isa portion suspected of having transcriptional activation or repressoractivity.
 14. The method of claim 6, wherein the transcriptionalmodulating portion is a portion suspected of having nuclear importactivity.
 15. The method of claim 6, wherein the transcriptionalmodulating portion is a portion suspected of having nuclear exportactivity.
 16. The method of claim 6, wherein the test compound comprisesthe selected ligand covalently linked to a transcriptional modulatingportion.
 17. The method of claim 6, wherein the test compound is amember of a combinatorial library.
 18. The method of claim 6, whereinthe changes in transcriptional activity are detected as variations inobserved levels of mRNA, or protein product encoded by the target gene.19. The method of claim 6, wherein the target gene is selected from thegroup consisting of a gene encoding a protein conferring resistance to adrug, a gene encoding an enzyme, a gene which rescues an auxotrophicphenotype, and a gene encoding a cell surface antigen.
 20. The method ofclaim 6, wherein the target gene encodes a protein which provides forcalorimetric, luminescent or fluorescent detection.
 21. The method ofclaim 6, wherein an increase in target gene activity or expression isindicative of a transcriptional activator.
 22. The method of claim 6,wherein a decrease in target gene activity or expression is indicativeof a transcriptional repressor.
 23. The method of claim 6, wherein thetranscriptional modulator is not itself the product of genetranscription or translation.
 24. The method of claim 6, wherein thetranscriptional modulator is membrane-permeant.
 25. The method of claim6, wherein the transcriptional modulator has a molecular weight of lessthan about 3 kDa.
 26. The method of claim 6, wherein the selected ligandis selected from the group consisting of FK506, FK520, rapamycin,cyclosporin A, tetracycline, steroid, and derivatives thereof which aremodified to have a higher binding affinity for the ligand-binding domaincompared to unmodified forms.
 27. The method of claim 6, wherein theligand-binding domain of the chimeric protein comprises between about 50and 350 amino acids.
 28. The method of claim 6, wherein theligand-binding domain of the chimeric protein is less than about 200amino acid residues in length.
 29. The method of claim 6, wherein theligand-binding domain of the chimeric protein binds to the ligand withan affinity of less than or equal to about 10⁻⁶ M.
 30. The method ofclaim 6, wherein the ligand-binding domain of the chimeric proteincomprises an immunophilin domain, a cyclophilin domain, a steroidbinding domain, an antibiotic domain, an antibody domain, adihydrofolate reductase (DHFR) domain, or a DNA gyrase domain.
 31. Themethod of claim 6, wherein the ligand-binding domain comprises an FK506binding protein ("FKBP") 12 or a variant thereof which is modified tohave a higher binding affinity for the selected ligand compared to anunmodified form.
 32. The method of claim 6, wherein the ligand-bindingdomain comprises an FK506 binding protein 12 variant which comprises upto 10 amino acid substitutions relative to wild-type FKBP.
 33. Themethod of claim 6, wherein the ligand-binding domain comprises an FK506binding protein 12 variant which comprises one or more substitutions ofTyr 26, Phe 36, Asp 37, Tyr 82 and Phe 99 with a different amino acid.34. The method of claim 6, wherein the DNA-binding domain is selectedfrom the group consisting of a homeodomain, and a zinc finger domain.35. The method of claim 31, wherein the FKBP12 is encoded by anucleotide sequence which selectively hybridizes to a nucleotidesequence encoding an FKBP or a variant thereof.